Deprotection and purification of oligonucleotides and their derivatives

ABSTRACT

Method for synthesis, deprotection, and/or purification of nucleic acid molecules, such as oligonucleotides comprising one or more ribonucleotides. Such nucleic acid molecules include siRNA, dsRNA, ribozymes, antisense, and aptamers.

This application is a Continuation-in-part of International ApplicationNo. PCT/US03/21775 filed Jul. 14, 2003, which is a Continuation-in-partof U.S. patent application Ser. No. 10/194,875, filed Jul. 12, 2002.These applications are hereby incorporated by reference herein in itsentirety, including the drawings.

BACKGROUND OF THE INVENTION

This invention relates to the synthesis, deprotection, and purificationof molecules comprising one or more ribonucleotides.

The following discussion relates to the synthesis, deprotection, andpurification of oligonucleotides containing one or more ribonucleotides.The discussion is not meant to be complete and is provided only forunderstanding the invention that follows. The summary is not anadmission that any of the work described below is prior art to theclaimed invention.

Research in the many roles of ribonucleic acids has, in the past, beenhindered by limited means of producing such biologically relevantmolecules (Cech, 1992, Nucleic Acids Research, 17, 7381-7393; Francklynand Schimmel, 1989, Nature, 337, 478-481; Cook et al., 1991, NucleicAcids Research, 19, 1577-1583; Gold, 1988, Annu. Rev. Biochemistry, 57,199-233). Although enzymatic methods existed, protocols that allowed oneto probe structure function relationships were limited. Only uniformpost-synthetic chemical modification (Karaoglu and Thurlow, 1991,Nucleic Acids Research, 19, 5293-5300) or site directed mutagenesis(Johnson and Benkovic, 1990, The Enzymes, Vol. 19, Sigman and Boyer,eds., 159-211) were available. In the latter case, researchers werelimited to usage of natural bases. Fortunately, adaptation of thephosphoramidite protocol for DNA synthesis to RNA synthesis has greatlyaccelerated our understanding of RNA. Site-specific introduction ofmodified nucleotides to any position in a given RNA has now becomeroutine. Furthermore, one is not confined to a single modification butcan include many variations in each molecule.

It is seemingly out of proportion that one small structural modificationcould cause such a dilemma. However, the presence of a single hydroxylat the 2′-position of the ribofuranose ring, has been the major reasonthat research in the RNA field has lagged so far behind comparable DNAstudies. Progress has been made in improving methods for DNA synthesisthat have enabled the production of large amounts of antisensedeoxyoligonucleotides for structural and therapeutic applications. Onlyrecently have similar gains been achieved for ribonucleotides (Wincottet al., 1995, Nucleic Acids Research, 23, 2677-2684; Sproat et al.,1995, Nucleosides and Nucleotides, 14, 255-273; Vargeese et al., 1998,Nucleic Acids Research, 26, 1046-1050).

The chasm between DNA and RNA synthesis is due to the difficulty ofidentifying orthogonal protecting groups for the 5′- and 2′-hydroxyls.Historically, two standard approaches have been taken by scientistsattempting to solve the RNA synthesis problem; developing a method thatis compatible with state-of the-art DNA synthesis or designing anapproach specifically suited for RNA. Although adaptation of the DNAprocess provides a more universal procedure in which non-RNAphosphoramidites can easily be incorporated into RNA oligomers, theadvantage to the latter approach is that one can develop a process thatis best for RNA synthesis and as a result, better yields can berealized. However, in both cases similar issues are faced, for exampleidentifying protecting groups that are compatible with synthesisconditions yet can be removed at the appropriate juncture. This problemdoes not refer only to the 2′- and 5′-OH groups, but includes the baseand phosphate protecting groups as well. Consequently, the accompanyingdeprotection steps, in addition to the choice of ancillary agents, areimpacted. Another shared issue is the need for efficient synthesis ofthe monomer building blocks.

Solid phase synthesis of oligoribonucleotides follows the same pathwayas DNA synthesis. A solid support with an attached nucleoside issubjected to removal of the protecting group on the 5′-hydroxyl. Theincoming phosphoramidite is coupled to the growing chain in the presenceof an activator. Any unreacted 5′-hydroxyl is capped and the phosphitetriester is then oxidized to provide the desired phosphotriesterlinkage. The process is then repeated until an oligomer of the desiredlength results. The actual reagents used may vary according to the 5′-and 2′-protecting groups. Other ancillary reagents may also differ.

Once the oligoribonucleotide has been synthesized, it must then bedeprotected. This is typically a two-step process that entails cleavageof the oligomer from the support and deprotection of the base andphosphate blocking groups, followed by removal of the 2′-protectinggroups. Occasionally, a different order of reactions or separatedeprotection of the phosphate groups is required. In all cases, it isimperative that indiscriminate removal of protecting groups not occur,this is particularly an issue in the classic situation wherein the firststep is base mediated. In this case, if the 2′-hydroxyl is revealedunder these conditions, strand scission will result due to attack of thevicinal hydroxyl group on the neighboring phosphate backbone. Two otherconcerns that are prevalent in RNA synthesis but play no part in DNA arethe propensity for 3′-2′ phosphodiester migration to provide undesired2′-5′ linkages and the susceptibility of oligoribonucleotides todegradation by ribonucleases. The latter fact has led many researchersto develop 2′-protecting groups that can remain in place until theoligomer is required for the desired experiment.

In the past, deprotection of oligoribonucleotides containing a2′-O-TBDMS (t-butyldimethylsilyl) group was a two step process thatfirst entailed a basic step similar to that used for the deprotection ofDNA in which the oligomer was cleaved from the support and the base andphosphate groups were removed. The initial step was accomplished in 1-4h at 55° C. with 3/1 NH₄OH/EtOH. Since the oligomer is not exposed tosevere deprotection conditions for prolonged periods, better yields ofhigher quality product result. More recently, a faster, two step,deprotection protocol, entailing the use of aqueous methylamine has beenreported for RNA (Usman et al., U.S. Pat. No. 5,804,683; Wincott et al.,1995, supra; Reddy et al., 1995, Tetrahedron Lett., 36, 8929-8932).Incubation times have been reduced to 10 min at 65° C. When comparedwith other RNA deprotection methods, treatment with this reagent gavegreater full length product than the standard protocol using 3/1NH₄OH/EtOH (Wincott et al., 1995, supra). The only requirement is thatacetyl must be used as the N-protecting group for cytidine because of awell-documented transamination reaction (Reddy et al., 1994, TetrahedronLett., 35, 4311-4314). As stated earlier, through the use of methylaminethis step has been reduced to 10 minutes. The second step is removal ofthe 2′-silyl protecting group from the oligonucleotide. In the past thishad been accomplished with 1 M n-tetrabutyl ammonium fluoride (TBAF) inTHF at room temperature over 24 h (Usman et al., 1987, J. Am. Chem.Soc., 109, 7845-7854; Scaringe et al., 1990, Nucleic Acids Research, 18,5433-5341). Unfortunately, the use of this deprotecting agent producessalts which must be removed prior to analysis and purification. Inaddition, the long exposure time required for complete removal of theprotecting group, coupled with the reagent's sensitivity to adventitiouswater (Hogrefe et al., 1994, Nucleic Acids Research, 21, 4739-4741),made it a less than ideal reagent. Although some reports have beenpublished regarding the use of neat triethylamine trihydrofluoride(TEA.3HF) (Duplaa et al., U.S. Pat. No. 5,552,539, Gasparutto et al.,1992, Nucleic Acids Research, 20, 5159-5166; Westman et al., 1994,Nucleic Acids Research, 22, 2430-2431) as a desilylating reagent,results have been mixed. A cocktail of TEA.3HF in combination withN-methyl pyrrolidinone (NMP) (Usman and Wincott, U.S. Pat. No.5,831,071; Wincott et al., 1995, supra) or DMF (Sproat et al., 1995,supra) has also been described in which full deprotection can beachieved in 30-90 min at 65° C. or 4-8 h at room temperature. As anadded advantage, since no salts are produced, the product can bedirectly precipitated from the desilylating reagent.

Tracz, U.S. Pat. No. 5,977,343; Tracz, U.S. Pat. No 5,686,599, describesa one-pot protocol for ribonucleotide deprotection using anhydrousmethylamine and triethylamine trihydrogen fluoride. This procedureinvolves the use of anhydrous methylamine followed by neat triethylaminetrihydrofluoride to effectively deprotect oligoribonucleotides in aone-pot fashion. However such a protocol may be cumbersome fordeprotection of oligonucleotides synthesized on a plate format, such asa 96-well plate, because it may be necessary to separate thesolid-support from the partially deprotected oligonucleotide prior tothe 2′-hydroxyl deprotection. Also, since the methylamine solution usedis anhydrous, it may be difficult to solubilize the negatively chargedoligoribonucleotides obtained after basic treatment. More recently thisprocedure has been reported in which both the basic deprotection and thedesilylation reaction can be accomplished in one-pot using a mixture ofanhydrous methylamine in ethanol followed by addition of TEA-3HF(Bellon, 1999, Current Protocols in Nucleic Acid Chemistry, Beaucage,Bergstrom, Glick and Jones, eds., in press). This protocol allows forthe complete deprotection of an oligoribonucleotide in less than 2 hwithout any evidence of 3′-2′ migration.

The parameters of 2′-deprotection are dictated by the correspondingprotecting groups utilized for differing 2′-chemistries present within agiven oligonucleotide. The use of alternate 2′-ribofuranosyl carbocyclefunctions within the same oligonucleotide molecule can present potentialproblems with respect to the synthesis, deprotection, and purificationof such molecules. The efficient synthesis of nucleic acids which arechemically modified to increase nuclease resistance while maintainingcatalytic activity is of importance to the potential development of newtherapeutic agents. Recently, Beaudry et al., 2000, Chemistry andBiology, 7, in press, describe the in vitro selection of a novelnuclease-resistant RNA phosphodiesterase. This enzymatic nucleic acidmolecule can contain both ribo (2′-hydroxyl) and amino(2′-deoxy-2′-amino) functions. The large scale synthesis ofoligonucleotides with both ribo and amino functions presents practicalproblems with regard to the concomitant removal oftert-Butyldimethylsilyl (TBDMSi) and N-phthaloyl protecting groups,while at the same time preserving the integrity of the ribonucleotidelinkages. The use of the N-phthaloyl protecting group for the 2′-aminogroup during oligonucleotide synthesis offers the benefit of improvedsynthetic yields compared to the trifluoroacetyl (TFA) and FMOC groups(Usman et al., U.S. Pat. No. 5,631,360; Beigelman et al., 1995, NucleicAcids Research, 23(21), 4434-4442). The phthaloyl group is readilycleaved with aqueous methylamine at 65° C. and the TBDMSi group isreadily cleaved using a fluoride ion source, such as tetrabutylammoniumfluoride (TBAF) or triethylammonium trihydrofluoride (TEA.3HF).Application of the “one pot” deprotection procedures described aboveresults in the incomplete deprotection of N-phthaloyl protection. Thetwo step deprotection procedure can be employed for the completedeprotection of oligonucleotides containing both ribo (2′-TBDMS) andamino (N-phthaloyl) protecting groups, however, this process is notreadily amenable to large scale oligonucleotide synthesis or multiwellplate oligonucleotide synthesis.

As such there exists an unmet need for a fast, efficient method whichallows for the complete deprotection of molecules containing both aminoand ribo carbohydrate moieties. Such a method will enable the largescale synthesis of such molecules for use as therapeutic agents and thesmall scale synthesis of such molecules for combinatorial screening.

SUMMARY OF THE INVENTION

Current oligonucleotide deprotection methods for oligonucleotidescomprising one or more ribonucleotides are limited by both the length oftime needed for complete deprotection and by the incomplete deprotectionof certain protecting groups (for example N-phthaloyl). The use ofanhydrous methylamine and triethylamine trihydrofluoride as a “one pot”deprotection cocktail makes use of DMSO to solubilize the partiallydeprotected oligonucleotide under anhydrous conditions (Tracz, U.S. Pat.No. 5,977,343). The use of aqueous methylamine has been avoided incombination with triethylamine trihydrofluoride up to this point due tothe presumed susceptibility of ribonucleotide linkages to degradationunder these conditions (for example, see example 3 described herein) asa result of alkaline hydrolysis (Brown et al., 1952, J. Chem. Soc.,London, 2708). This has been overcome with the separation of the aqueousmethylamine treatment from the triethylamine trihydrofluoride treatmentby making use of an intermediary drying step to remove the aqueousmethylamine reagent prior to removal of the 2′-hydoxyl protecting group,thereby precluding alkaline hydrolysis of the ribonucleotide linkages.This two step process is not amenable to large scale oligonucleotidesynthesis and oligonucleotide synthesis performed on a multi-well plate,high throughput format. The use of a “one-pot” deprotection methodcomprising treatment with anhydrous methylamine and triethylaminetrihydrofluoride in the presence of DMSO as a co-solvent is benign toribonucleotide linkages, however, this process may require additionaloptimization in terms of both total deprotection time and resultingoligonucleotide quality. In addition, the “one-pot” anhydrous method isnot very effective for the complete removal of some protecting groups(for example N-phthaloyl). The deprotection method of the instantinvention provides a rapid, “one-pot” method for the completedeprotection of oligonucleotides comprising one or more ribonucleotides,and is further capable of complete deprotection of a wide variety ofoligonucleotide protecting groups, including the N-phthaloyl group.

This invention concerns a process for the deprotection and purificationof molecules comprising one or more ribonucleotides. Specifically, thepresent invention features a method for the removal of protecting groupsfrom nucleic acid base, phosphate, and 2′-hydroxyl (2′-OH) and/or2′-deoxy-2′-amino (2′-NH₂) groups, which allows the deprotection andsubsequent purification of molecules comprising one or moreribonucleotides in both a large scale and a high throughput manner.

In a preferred embodiment, the invention features a one-pot process forrapid deprotection of molecules comprising one or more ribonucleotides.In additional embodiments, the instant invention features a process forthe rapid deprotection of molecules comprising ribonucleotides and/or2′-deoxy-2′-amino ribofuranose moieties which are protected withalkylsilyl and/or phthaloyl-based protecting groups respectively.Specifically, the invention provides a process for the rapiddeprotection of molecules comprising both ribonucleotides and/or2′-deoxy-2′-amino ribofuranose moieties which are protected witht-butyldimethylsilyl (TBDMSi) and/or N-phthaloyl protecting groupsrespectively

In preferred embodiments, the instant invention features the use of anaqueous methylamine solution to partially deprotect molecules comprisingone or more ribonucleotides followed by treatment with triethylammoniumtrihydrofluoride for the complete deprotection of molecules. In anotherembodiment, the treatment with triethylammonium trihydrofluoride is inthe presence of a co-solvent (for example, DMSO).

In one embodiment, the invention features a process for the synthesis,deprotection, and purification of molecules comprising one or moreribonucleotides, comprising the steps of: (a) solid phase, solutionphase, and/or hybrid phase, (e.g.; phosphoramidite-based orH-phosphonate-based) oligonucleotide synthesis comprising the steps ofdetritylation, activation, coupling, capping, and oxidation or theequivalent thereof, in any suitable order, followed by (b) deprotectioncomprising contacting the nucleic acid molecule having one or moreribonucleotides with aqueous alkylamine (where alkyl can be ethyl,propyl or butyl and is preferably methyl, e.g.; methylamine, for example40% aqueous methylamine), and/or trialkylamine (where alkyl can bemethyl, propyl or butyl and is preferably ethyl, e.g.; triethylamine) atabout 10 to about 100° C., or about 20° C. to about 80° C., or about 30°C. to about 65° C., preferably about 35° C. or about 65° C. formolecules comprising N-phthaloyl protecting groups, for about 5 to about240 minutes, about 20 to about 100 minutes, preferably about 60 minutesunder conditions suitable for partial deprotection of theoligonucleotide, and contacting the partially deprotected moleculecomprising one or more ribonucleotides withtriethylamine.trihydrofluoride (TEA.3HF) in the presence of a solvent(for example DMSO, DMF, HMPA, ethanol, methanol, isopropanol,N-methylpyrrolidinone and others) and heating at about about 10 to about100° C., preferably at about 65° C., for about 5 to about 240 minutes,preferably about 60 minutes, to remove 2′-hydroxyl protecting groups(for example, t-butyldimethylsilyl), then quenching the deprotectionreaction by using aqueous sodium acetate, ammonium bicarbonate, and/ortriethylammonium bicarbonate or the equivalent thereof, preferably 50 mMaqueous sodium acetate, then (c) purifying the molecule comprising oneor more ribonucleotides, comprising loading the deprotected productsonto media comprising Pharmacia Source Q15 and Biorad Macroprep 25Qmedia, or the equivalent thereof such as Pharmacia Q-sepharose,Perceptive POROS HQ, TOSOHAAS Q-5PW-HR, Q-5PW, or super Q-5PW,equilibrated with a buffer comprising either 20% ethanol or acetonitrilein about 20 mM sodium phosphate and about 0.1 M NaCl, in a loadingbuffer comprising water, or either 20% ethanol or acetonitrile in about20 mM sodium phosphate and about 0.1 M NaCl, and applying a suitablegradient of about 1.0 M NaCl as an elution buffer, then analyzing thefractions by a suitable technique and allowing for the pure fractions tobe pooled and desalted via tangential flow filtration or the equivalentthereof, by using membranes comprising such membranes as those selectedfrom the group consisting of Sartorius or Pall Filtron PES 1 Kmembranes, then lyophilizing the concentrated material.

In one embodiment, the invention features a process for the synthesis,deprotection, and purification of a nucleic acid molecule comprising oneor more ribonucleotides and one or more chemical modifications,comprising the steps of: (a) solid phase, solution phase, and/or hybridphase, (e.g.; phosphoramidite-based or H-phosphonate-based)oligonucleotide synthesis comprising the steps of detritylation,activation, coupling, capping, and oxidation or the equivalent thereof,in any suitable order, followed by (b) deprotection comprisingcontacting the nucleic acid molecule with aqueous alkylamine (wherealkyl can be ethyl, propyl or butyl and is preferably methyl, e.g.;methylamine, for example 40% aqueous methylamine), and/or trialkylamine(where alkyl can be methyl, propyl or butyl and is preferably ethyl,e.g.; triethylamine) at about 10 to about 100° C., or about 20° C. toabout 80° C., or about 30° C. to about 65° C., preferably about 35° C.or about 65° C. for molecules comprising N-phthaloyl protecting groups,for about 5 to about 240 minutes, about 20 to about 100 minutes,preferably about 60 minutes under conditions suitable for partialdeprotection of the nucleic acid molecule, and contacting the partiallydeprotected molecule comprising one or more ribonucleotides withtriethylamine.trihydrofluoride (TEA.3HF) in the presence of a solvent(for example DMSO, DMF, HMPA, ethanol, methanol, isopropanol,N-methylpyrrolidinone and others) and heating at about about 10 to about100° C., preferably at about 65° C., for about 5 to about 240 minutes,preferably about 60 minutes, to remove 2′-hydroxyl protecting groups(for example, t-butyldimethylsilyl), then quenching the deprotectionreaction by using aqueous sodium acetate, ammonium bicarbonate, and/ortriethylammonium bicarbonate or the equivalent thereof, preferably 50 mMaqueous sodium acetate, then (c) purifying the nucleic acid molecule,comprising loading the deprotected products onto media comprisingPharmacia Source Q15 and Biorad Macroprep 25Q media, or the equivalentthereof such as Pharmacia Q-sepharose, Perceptive POROS HQ, TOSOHAASQ-5PW-HR, Q-5PW, or super Q-5PW, equilibrated with a buffer comprisingeither 20% ethanol or acetonitrile in about 20 mM sodium phosphate andabout 0.1 M NaCl, in a loading buffer comprising water, or either 20%ethanol or acetonitrile in about 20 mM sodium phosphate and about 0.1 MNaCl, and applying a suitable gradient of about 1.0 M NaCl as an elutionbuffer, then analyzing the fractions by a suitable technique andallowing for the pure fractions to be pooled and desalted via tangentialflow filtration or the equivalent thereof, by using membranes comprisingsuch membranes as those selected from the group consisting of Sartoriusor Pall Filtron PES 1 K membranes, then lyophilizing the concentratedmaterial. In another embodiment, the chemical modifications, which maybe same or different, independently include sugar modifications, such as2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-C-alkyl, 2′-deoxy, 2′-deoxy-2′-aminoor LNA (locked nucleic acid) nucleosides. In another embodiment, thechemical modifications in the nucleic acid molecule, which may be sameor different, includes one or more phosphorothioate internucleotidelinkages. In another embodiment, the chemical modification of nucleicacid molecule, which may be same or different, comprises modifying theterminal positions with one or more abasic moieties. In anotherembodiment, the chemically modified nucleic acid molecule comprises aterminal cap moiety at the 3′-end, 5′-end, or both 3′ and 5′ ends of theoligonucleotide.

In another embodiment the nucleic acid molecules synthesized,deprotected and/or purified according to the invention comprise acombination of sugar, phosphate backbone, base, and/or terminal endmodifications.

In one embodiment, the invention features a process for the deprotectionand subsequent purification of nucleic acid molecules having one or moreribonucleotides with protecting groups, comprising the steps of: (a)deprotection comprising contacting the nucleic acid molecule withaqueous alkylamine (where alkyl can be ethyl, propyl or butyl and ispreferably methyl, e.g.; methylamine, for example 40% aqueousmethylamine), and/or trialkylamine (where alkyl can be methyl, propyl orbutyl and is preferably ethyl, e.g.; triethylamine) at about 10 to about100° C., or about 20° C. to about 80° C., or about 30° C. to about 65°C., preferably about 35° C. or about 65° C. for molecules comprisingN-phthaloyl protecting groups, for about 5 to about 240 minutes, orabout 20 to about 100 minutes, preferably about 60 minutes underconditions suitable for partial deprotection of the oligonucleotide, andcontacting the partially deprotected molecule withtriethylamine.trihydrofluoride (TEA.3HF) in the presence of a solvent(for example DMSO, DMF, HMPA, ethanol, methanol, isopropanol,N-methylpyrrolidinone and others) and heating at about 10 to about 100°C., preferably at about 65° C., for about 5 to about 240 minutes,preferably about 60 minutes, to remove 2′-hydroxyl protecting groups(for example, t-butyldimethylsilyl), then quenching the deprotectionreaction by using aqueous sodium acetate, ammonium bicarbonate, and/ortriethylammonium bicarbonate or the equivalent thereof, preferably 50 mMaqueous sodium acetate, then (b) purifying the molecule comprising oneor more ribonucleotides, comprising loading the deprotection productsonto media comprising Pharmacia Source Q15 and Biorad Macroprep 25Qmedia, or the equivalent thereof, such as Pharmacia Q-sepharose,Perceptive POROS HQ, TOSOHAAS Q-5PW-HR, Q-5PW, or super Q-5PW,equilibrated with a buffer comprising either 20% ethanol or acetonitrilein about 20 mM sodium phosphate and about 0.1 M NaCl, in a loadingbuffer comprising water, or either 20% ethanol or acetonitrile in about20 mM sodium phosphate and about 0.1 M NaCl, and applying a suitablegradient of about 1.0 M NaCl as an elution buffer, then analyzing thefractions by a suitable technique and allowing for the pure fractions tobe pooled and desalted via tangential flow filtration or the equivalentthereof, by using membranes comprising such membranes as those selectedfrom the group consisting of Sartorius or Pall Filtron PES 1 Kmembranes, then lyophilizing the concentrated material.

In one embodiment, the invention features a process for the deprotectionand subsequent purification of nucleic acid molecules having one or moreribonucleotides one or more chemical modifications, and with protectinggroups, comprising the steps of: (a) deprotection comprising contactingthe nucleic acid molecule with aqueous alkylamine (where alkyl can beethyl, propyl or butyl and is preferably methyl, e.g.; methylamine, forexample 40% aqueous methylamine), and/or trialkylamine (where alkyl canbe methyl, propyl or butyl and is preferably ethyl, e.g.; triethylamine)at about 10 to about 100° C., or about 20° C. to about 80° C., or about30° C. to about 65° C., preferably about 35° C. or about 65° C. fornucleic acid molecules comprising N-phthaloyl protecting groups, forabout 5 to about 240 minutes, or about 20 to about 100 minutes,preferably about 60 minutes under conditions suitable for partialdeprotection of the nucleic acid molecule, and contacting the partiallydeprotected molecule with triethylamine.trihydrofluoride (TEA.3HF) inthe presence of a solvent (for example DMSO, DMF, HMPA, ethanol,methanol, isopropanol, N-methylpyrrolidinone and others) and heating atabout 10 to about 100° C., preferably at about 65° C., for about 5 toabout 240 minutes, preferably about 60 minutes, to remove 2′-hydroxylprotecting groups (for example, t-butyldimethylsilyl), then quenchingthe deprotection reaction by using aqueous sodium acetate, ammoniumbicarbonate, and/or triethylammonium bicarbonate or the equivalentthereof, preferably 50 mM aqueous sodium acetate, then (b) purifying thenucleic acid molecule, comprising loading the deprotection products ontomedia comprising Pharmacia Source Q15 and Biorad Macroprep 25Q media, orthe equivalent thereof, such as Pharmacia Q-sepharose, Perceptive POROSHQ, TOSOHAAS Q-5PW-HR, Q-5PW, or super Q-5PW, equilibrated with a buffercomprising either 20% ethanol or acetonitrile in about 20 mM sodiumphosphate and about 0.1 M NaCl, in a loading buffer comprising water, oreither 20% ethanol or acetonitrile in about 20 mM sodium phosphate andabout 0.1 M NaCl, and applying a suitable gradient of about 1.0 M NaClas an elution buffer, then analyzing the fractions by a suitabletechnique and allowing for the pure fractions to be pooled and desaltedvia tangential flow filtration or the equivalent thereof, by usingmembranes comprising such membranes as those selected from the groupconsisting of Sartorius or Pall Filtron PES 1 K membranes, thenlyophilizing the concentrated material. In another embodiment, thechemically modified nucleic acid molecule comprises independently one ormore 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-C-alkyl, 2′-deoxy,2′-deoxy-2′-amino or LNA (locked nucleic acid) nucleosides. In anotherembodiment, the chemically modified nucleic acid molecule comprises oneor more phosphorothioate internucleotide linkages.

In another embodiment, the chemically modified nucleic acid moleculecomprises one or more abasic moieties. In another embodiment, thechemically modified nucleic acid molecule comprises a terminal capmoiety at the 3′-end, 5′-end, or both 3′ and 5′ ends of theoligonucleotide.

In yet another preferred embodiment, the invention features a processfor one pot deprotection of nucleic acid molecules having one or moreribonucleotides with protecting groups, comprising the steps of: (a)contacting the nucleic acid molecule with aqueous alkylamine (wherealkyl can be ethyl, propyl or butyl and is preferably methyl, e.g.;methylamine, for example 40% aqueous methylamine), and/or trialkylamine(where alkyl can be methyl, propyl or butyl and is preferably ethyl,e.g.; triethylamine) at about 10 to 100° C., or about 20° C. to about80° C., or about 30° C. to about 65° C., preferably about 35° C. orabout 65° C. for molecules comprising N-phthaloyl protecting groups, forabout 5 to about 240 minutes, or about 20 to about 100 minutes,preferably about 60 minutes, under conditions suitable for partialdeprotection of the oligonucleotide, and (b) contacting the partiallydeprotected molecule with triethylamine.trihydrofluoride (TEA.3HF) inthe presence of a solvent (for example DMSO DMF, HMPA, ethanol,methanol, isopropanol, N-methylpyrrolidinone and others) and heating atabout 10 to 100° C., preferably at about 65° C., for about 5 to 240minutes, preferably about 60 minutes, to remove 2′-hydroxyl protectinggroups (for example, t-butyldimethylsilyl). In additional embodiments,other alkylamine.HF complexes may also be used, (e.g.; trimethylaminetrihydrofluoride and/or diisopropylethylamine trihydrofluoride) underconditions suitable for the complete deprotection of the molecule.

In yet another preferred embodiment, the invention features a processfor one pot deprotection of nucleic acid molecules having one or moreribonucleotides, one or more chemical modifications, and with protectinggroups, comprising the steps of: (a) contacting the nucleic acidmolecule with aqueous alkylamine (where alkyl can be ethyl, propyl orbutyl and is preferably methyl, e.g.; methylamine, for example 40%aqueous methylamine), and/or trialkylamine (where alkyl can be methyl,propyl or butyl and is preferably ethyl, e.g.; triethylamine) at about10 to 100° C., or about 20° C. to about 80° C., or about 30° C. to about65° C., preferably about 35° C. or about 65° C. for molecules comprisingN-phthaloyl protecting groups, for about 5 to about 240 minutes, orabout 20 to about 100 minutes, preferably about 60 minutes, underconditions suitable for partial deprotection of the nucleic acidmolecule, and (b) contacting the partially deprotected molecule withtriethylamine.trihydrofluoride (TEA.3HF) in the presence of a solvent(for example DMSO DMF, HMPA, ethanol, methanol, isopropanol,N-methylpyrrolidinone and others) and heating at about 10 to 100° C.,preferably at about 65° C., for about 5 to 240 minutes, preferably about60 minutes, to remove 2′-hydroxyl protecting groups (for example,t-butyldimethylsilyl). In additional embodiments, other alkylamine.HFcomplexes may also be used, (e.g.; trimethylamine trihydrofluorideand/or diisopropylethylamine trihydrofluoride) under conditions suitablefor the complete deprotection of the molecule. In another embodiment,the chemically modified nucleic acid molecule comprises independentlyone or more 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-C-alkyl, 2′-deoxy,2′-deoxy-2′-amino or LNA (locked nucleic acid) nucleosides. In anotherembodiment, the chemically modified nucleic acid molecule comprises oneor more phosphorothioate internucleotide linkages. In anotherembodiment, the chemically modified nucleic acid molecule comprises oneor more abasic moieties. In another embodiment, the chemically modifiednucleic acid molecule comprises a terminal cap moiety at the 3′-end,5′-end, or both 3′ and 5′ ends of the oligonucleotide.

In a preferred embodiment, the invention features a process forpurifying a nucleic acid molecule, comprising the steps of: (a) loadingthe crude deprotected molecule onto media comprising Pharmacia SourceQ15 and Biorad Macroprep 25Q media, or the equivalent thereof such asPharmacia Q-sepharose, Perceptive POROS HQ, TOSOHAAS Q-5PW-HR, Q-5PW, orsuper Q-5PW, equilibrated with a buffer comprising either 20% ethanol oracetonitrile in about 20 mM sodium phosphate and about 0.1 M NaCl, in aloading buffer comprising water, or either 20% ethanol or acetonitrilein about 20 mM sodium phosphate and about 0.1 M NaCl, and (b) applying asuitable gradient of about 1.0 M NaCl as an elution buffer, thenanalyzing the fractions by a suitable technique and allowing for thepure fractions to be pooled and desalted via tangential flow filtrationor the equivalent thereof, by using membranes comprising such membranesas those selected from the group consisting of Sartorius or Pall FiltronPES 1 K membranes.

In a preferred embodiment, the invention features a process forpurifying a chemically modified nucleic acid molecule, comprising thesteps of: (a) loading the crude deprotected molecule onto mediacomprising Pharmacia Source Q15 and Biorad Macroprep 25Q media, or theequivalent thereof such as Pharmacia Q-sepharose, Perceptive POROS HQ,TOSOHAAS Q-5PW-HR, Q-5PW, or super Q-5PW, equilibrated with a buffercomprising either 20% ethanol or acetonitrile in about 20 mM sodiumphosphate and about 0.1 M NaCl, in a loading buffer comprising water, oreither 20% ethanol or acetonitrile in about 20 mM sodium phosphate andabout 0.1 M NaCl, and (b) applying a suitable gradient of about 1.0 MNaCl as an elution buffer, then analyzing the fractions by a suitabletechnique and allowing for the pure fractions to be pooled and desaltedvia tangential flow filtration or the equivalent thereof, by usingmembranes comprising such membranes as those selected from the groupconsisting of Sartorius or Pall Filtron PES 1 K membranes. In anotherembodiment, the chemically modified nucleic acid molecule comprisesindependently one or more 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-C-alkyl,2′-deoxy, 2′-deoxy-2′-amino or LNA (locked nucleic acid) nucleosides. Inanother embodiment, the chemically modified nucleic acid moleculecomprises one or more phosphorothioate internucleotide linkages. Inanother embodiment, the chemically modified nucleic acid moleculecomprises one or more abasic moieties. In another embodiment, thechemically modified nucleic acid molecule comprises a terminal capmoiety at the 3′-end, 5′-end, or both 3′ and 5′ ends of theoligonucleotide.

In an additional preferred embodiment, the nucleic acid molecule orchemically modified nucleic acid molecule is lyophilized afterpurification.

In preferred embodiments, the deprotection reaction can be quenched byusing aqueous sodium acetate, ammonium bicarbonate, and/ortriethylammonium bicarbonate or the equivalent thereof, preferably 50 mMaqueous sodium acetate.

In another preferred embodiment, the invention features a process forthe deprotection of nucleic acid molecules comprising an oligonucleotidehaving 2′-N-phthaloyl and 2′-O-silyl protection comprising the steps of:(a) contacting the nucleic acid molecule with aqueous alkylamine (wherealkyl can be ethyl, propyl or butyl and is preferably methyl, e.g.;methylamine, for example 40% aqueous methylamine), and/or trialkylamine(where alkyl can be methyl, propyl or butyl and is preferably ethyl,e.g.; triethylamine) at about 10 to 100° C., or about 20° C. to about80° C., or about 30° C. to about 65° C., preferably about 35° C. orabout 65° C. for molecules comprising N-phthaloyl protecting groups, forabout 5 to about 240 minutes, or about 20 to about 100 minutes,preferably about 60 minutes, under conditions suitable for partialdeprotection of the oligonucleotide, and (b) contacting the partiallydeprotected molecule with triethylamine.trihydrofluoride (TEA.3HF) inthe presence of a solvent (for example DMSO DMF, HMPA, ethanol,methanol, isopropanol, N-methylpyrrolidinone and others) and heating atabout 10 to 100° C., preferably at about 65° C., for about 5 to 240minutes, preferably about 60 minutes, to remove 2′-hydroxyl protectinggroups (for example, t-butyldimethylsilyl). In additional embodiments,other alkylamine.HF complexes may also be used, (e.g.; trimethylaminetrihydrofluoride and/or diisopropylethylamine trihydrofluoride) underconditions suitable for the complete deprotection of the molecule. Thenucleic acid molecule can further comprise chemical modifications, suchas one or more 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-C-alkyl, 2′-deoxy,2′-deoxy-2′-amino or LNA (locked nucleic acid) nucleosides; one or morephosphorothioate internucleotide linkages; one or more abasic moieties;or a terminal cap moiety at the 3′-end, 5′-end, or both 3′ and 5′ endsof the oligonucleotide.

In a preferred embodiment, the partially deprotected molecule isfiltered using a suitable filtering medium, such as sintered glass, andwashed with a polar solvent (for example, DMSO, DMF, ethanol, methanol,isopropanol, and/or N-methylpyrrolidinone) prior to treatment withTEA.3HF reagent. In additional embodiments, the filtrate is cooled priorto treatment with TEA.3HF reagent, preferably to between about 0° C. and−78° C.

In another aspect the invention features a process for oligonucleotidedeprotection where the deprotection reaction is performed with theaqueous methylamine solution at temperatures ranging from about 0° C. to120° C. for a time of about 500 minutes to about 5 minutes.

In one embodiment, the invention features a process for deprotectingoligonucleotides including one or more 2′-deoxy-2′-fluoro nucleotidescomprising: contacting the oligonucleotide with a solution of aqueousmethylamine (e.g. 40% aqueous methylamine) at about 25° C. to about 45°C. for about 30 minutes. In another embodiment, the oligonucleotide iscontacted with the solution of aqueous methylamine at about 35° C. forabout 30 minutes.

In one embodiment, the invention features a process for deprotectingoligonucleotides including one or more 2′-deoxy-2′-fluoro nucleotidesand one or more ribonucleotides comprising: (a) contacting theoligonucleotide with a solution of aqueous methylamine (e.g. 40% aqueousmethylamine) at about 25° C. to about 45° C. for about 30 minutes, and(b) contacting the partially deprotected molecule of (a) withtriethylamine.trihydrofluoride (TEA.3HF) in the presence of a solvent(for example DMSO DMF, HMPA, ethanol, methanol, isopropanol,N-methylpyrrolidinone and others) and heating at about 10 to 100° C.,preferably at about 65° C., for an additional 5 to 60 minutes,preferably about 15 minutes, to remove 2′-hydroxyl protecting groups(for example, t-butyldimethylsilyl). In additional embodiments, otheralkylamine.HF complexes may also be used, (e.g.; trimethylaminetrihydrofluoride and/or diisopropylethylamine trihydrofluoride) underconditions suitable for the complete deprotection of the molecule. Inanother embodiment, the oligonucleotide in (a) is contacted with thesolution of aqueous methylamine at about 35° C. for about 30 minutes.

In one embodiment, a process for deprotection of molecules comprisingone or more ribonucleotides of the present invention is used todeprotect an oligonucleotide synthesized using a column format.

In one embodiment, a process for deprotection of molecules comprisingone or more ribonucleotides of the present invention is used todeprotect an oligonucleotide synthesized according to methods describedin Usman et al., U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203;6,117,657; 6,353,098; 6,362,323; 6,437,117; 6,469,158; and/or Vargeeseet al., U.S. Ser. No. 10/190,359, all incorporated by reference hereinin their entirety including the drawings.

In one embodiment, the invention features a method comprising: (a)synthesizing an oligonucleotide molecule having one or moreribonucleotides, (b) contacting the oligonucleotide with aqueousalkylamine (where alkyl can be ethyl, propyl or butyl and is preferablymethyl, e.g.; methylamine, for example 40% aqueous methylamine), and/ortrialkylamine (where alkyl can be methyl, propyl or butyl and ispreferably ethyl, e.g.; triethylamine) at about 10 to about 100° C., orabout 20° C. to about 80° C., or about 30° C. to about 65° C.,preferably 35° C. or 65° C. for about 15 to 240 minutes, preferablyabout 30 to about 60 minutes, under conditions suitable for partialdeprotection of the oligonucleotide, and (c) contacting the partiallydeprotected molecule with triethylamine.trihydrofluoride (TEA.3HF) inthe presence of a solvent (for example DMSO DMF, HMPA, ethanol,methanol, isopropanol, N-methylpyrrolidinone and others) and heating atabout 10 to about 100° C., preferably at about 65° C., for about 15 to240 minutes, preferably about 30 to about 60 minutes, to remove2′-hydroxyl protecting groups (for example, t-butyldimethylsilyl). Inadditional embodiments, other alkylamine.HF complexes may also be used,(e.g.; trimethylamine trihydrofluoride and/or diisopropylethylaminetrihydrofluoride) under conditions suitable for the completedeprotection of the molecule.

In one embodiment, the invention features a method comprising: (a)synthesizing an oligonucleotide molecule having one or moreribonucleotides, (b) contacting the oligonucleotide with aqueousalkylamine (where alkyl can be ethyl, propyl or butyl and is preferablymethyl, e.g.; methylamine, for example 40% aqueous methylamine), atabout 35° C. to about 65° C. for about 15 to about 240 minutes, (e.g.about 30 to about 60 minutes, under conditions suitable for partialdeprotection of the oligonucleotide, and (c) contacting the partiallydeprotected molecule with triethylamine.trihydrofluoride (TEA.3HF) inthe presence of a solvent (for example DMSO DMF, HMPA, ethanol,methanol, isopropanol, N-methylpyrrolidinone and others) and heating atabout 10 to 100° C., preferably at about 65° C., for about 15 to 240minutes, preferably about 60 minutes, to remove 2′-hydroxyl protectinggroups (for example, t-butyldimethylsilyl). In additional embodiments,other alkylamine.HF complexes may also be used, (e.g.; trimethylaminetrihydrofluoride and/or diisopropylethylamine trihydrofluoride) underconditions suitable for the complete deprotection of the molecule.

In one embodiment, the invention features a method comprising: (a)synthesizing an oligonucleotide molecule having one or more2′-deoxy-2′-fluoro nucleotides and one or more ribonucleotides, (b)contacting the oligonucleotide with aqueous alkylamine (where alkyl canbe ethyl, propyl or butyl and is preferably methyl, e.g.; methylamine,for example 40% aqueous methylamine), and/or trialkylamine (where alkylcan be methyl, propyl or butyl and is preferably ethyl, e.g.;triethylamine) at about 10 to about 100° C., or about 20° C. to about80° C., or about 30° C. to about 65° C., preferably 35° C. for about 15to 240 minutes, preferably about 30 minutes, under conditions suitablefor partial deprotection of the oligonucleotide, and (c) contacting thepartially deprotected molecule with triethylamine.trihydrofluoride(TEA.3HF) in the presence of a solvent (for example DMSO DMF, HMPA,ethanol, methanol, isopropanol, N-methylpyrrolidinone and others) andheating at about 10 to about 100° C., preferably at about 65° C., forabout 15 to 240 minutes, preferably about 15 to about 30 minutes, toremove 2′-hydroxyl protecting groups (for example,t-butyldimethylsilyl). In additional embodiments, other alkylamine.HFcomplexes may also be used, (e.g.; trimethylamine trihydrofluorideand/or diisopropylethylamine trihydrofluoride) under conditions suitablefor the complete deprotection of the molecule.

In one embodiment, the invention features a method comprising: (a)synthesizing an oligonucleotide molecule having one or more2′-deoxy-2′-fluoro nucleotides and one or more ribonucleotides, (b)contacting the oligonucleotide with aqueous alkylamine (where alkyl canbe ethyl, propyl or butyl and is preferably methyl, e.g.; methylamine,for example 40% aqueous methylamine), at about 30° C. to about 65° C.,preferably about 35° C. for about 15 to about 240 minutes, (e.g. about30 to about 60 minutes, under conditions suitable for partialdeprotection of the oligonucleotide, and (c) contacting the partiallydeprotected molecule with triethylamine.trihydrofluoride (TEA.3HF) inthe presence of a solvent (for example DMSO DMF, HMPA, ethanol,methanol, isopropanol, N-methylpyrrolidinone and others) and heating atabout 10 to 100° C., preferably at about 65° C., for about 15 to 240minutes, preferably about 15 to about 30 minutes, to remove 2′-hydroxylprotecting groups (for example, t-butyldimethylsilyl). In additionalembodiments, other alkylamine.HF complexes may also be used, (e.g.;trimethylamine trihydrofluoride and/or diisopropylethylaminetrihydrofluoride) under conditions suitable for the completedeprotection of the molecule.

In one embodiment, the invention features a method comprising: (a)synthesizing an oligonucleotide molecule having one or moreribonucleotides, (b) contacting the oligonucleotide with aqueousalkylamine (where alkyl can be ethyl, propyl or butyl and is preferablymethyl, e.g.; methylamine, for example 40% aqueous methylamine), and/ortrialkylamine (where alkyl can be methyl, propyl or butyl and ispreferably ethyl, e.g.; triethylamine) at about 10 to about 100° C., orabout 20° C. to about 80° C., or about 30° C. to about 65° C.,preferably 35° C. or 65° C. for about 15 to 240 minutes, preferablyabout 30 to about 60 minutes, under conditions suitable for partialdeprotection of the oligonucleotide, (c) contacting the partiallydeprotected molecule with triethylamine.trihydrofluoride (TEA.3HF) inthe presence of a solvent (for example DMSO DMF, HMPA, ethanol,methanol, isopropanol, N-methylpyrrolidinone and others) and heating atabout 10 to about 100° C., preferably at about 65° C., for about 15 to240 minutes, preferably about 30 to about 60 minutes, to remove2′-hydroxyl protecting groups (for example, t-butyldimethylsilyl, and(d), purifying the deprotected oligonucleotide under conditions suitablefor isolating the oligonucleotide.

In one embodiment, the invention features a method comprising: (a)synthesizing an oligonucleotide molecule having one or moreribonucleotides, (b) contacting the oligonucleotide with aqueousalkylamine (where alkyl can be ethyl, propyl or butyl and is preferablymethyl, e.g.; methylamine, for example 40% aqueous methylamine), atabout 35° C. to about 65° C. for about 15 to about 240 minutes, (e.g.about 30 to about 60 minutes, under conditions suitable for partialdeprotection of the oligonucleotide, and (c) contacting the partiallydeprotected molecule with triethylamine.trihydrofluoride (TEA.3HF) inthe presence of a solvent (for example DMSO DMF, HMPA, ethanol,methanol, isopropanol, N-methylpyrrolidinone and others) and heating atabout 10 to 100° C., preferably at about 65° C., for about 15 to 240minutes, preferably about 60 minutes, to remove 2′-hydroxyl protectinggroups (for example, t-butyldimethylsilyl), and (d), purifying thedeprotected oligonucleotide under conditions suitable for isolating theoligonucleotide.

In one embodiment, the invention features a method comprising: (a)synthesizing an oligonucleotide molecule having one or more2′-deoxy-2′-fluoro nucleotides and one or more ribonucleotides, (b)contacting the oligonucleotide with aqueous alkylamine (where alkyl canbe ethyl, propyl or butyl and is preferably methyl, e.g.; methylamine,for example 40% aqueous methylamine), and/or trialkylamine (where alkylcan be methyl, propyl or butyl and is preferably ethyl, e.g.;triethylamine) at about 10 to about 100° C., or about 20° C. to about80° C., or about 30° C. to about 65° C., preferably 35° C. for about 15to 240 minutes, preferably about 30 minutes, under conditions suitablefor partial deprotection of the oligonucleotide, and (c) contacting thepartially deprotected molecule with triethylamine.trihydrofluoride(TEA.3HF) in the presence of a solvent (for example DMSO DMF, HMPA,ethanol, methanol, isopropanol, N-methylpyrrolidinone and others) andheating at about 10 to about 100° C., preferably at about 65° C., forabout 15 to 240 minutes, preferably about 15 to about 30 minutes, toremove 2′-hydroxyl protecting groups (for example,t-butyldimethylsilyl). In additional embodiments, other alkylamine.HFcomplexes may also be used, (e.g.; trimethylamine trihydrofluorideand/or diisopropylethylamine trihydrofluoride) under conditions suitablefor the complete deprotection of the molecule, and (d), purifying thedeprotected oligonucleotide under conditions suitable for isolating theoligonucleotide.

In one embodiment, the invention features a method comprising: (a)synthesizing an oligonucleotide molecule having one or more2′-deoxy-2′-fluoro nucleotides and one or more ribonucleotides, (b)contacting the oligonucleotide with aqueous alkylamine (where alkyl canbe ethyl, propyl or butyl and is preferably methyl, e.g.; methylamine,for example 40% aqueous methylamine), at about 30° C. to about 65° C.,preferably about 35° C. for about 15 to about 240 minutes, (e.g. about30 to about 60 minutes, under conditions suitable for partialdeprotection of the oligonucleotide, and (c) contacting the partiallydeprotected molecule with triethylamine.trihydrofluoride (TEA.3HF) inthe presence of a solvent (for example DMSO DMF, HMPA, ethanol,methanol, isopropanol, N-methylpyrrolidinone and others) and heating atabout 10 to 100° C., preferably at about 65° C., for about 15 to 240minutes, preferably about 15 to about 30 minutes, to remove 2′-hydroxylprotecting groups (for example, t-butyldimethylsilyl). In additionalembodiments, other alkylamine.HF complexes may also be used, (e.g.;trimethylamine trihydrofluoride and/or diisopropylethylaminetrihydrofluoride) under conditions suitable for the completedeprotection of the molecule, and (d), purifying the deprotectedoligonucleotide under conditions suitable for isolating theoligonucleotide.

In one embodiment, an oligonucleotide of the invention is chemicallymodified. Non-limiting examples of chemically modifications include2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-C-alkyl, 2′-deoxy, 2′-deoxy-2′-aminoor LNA (locked nucleic acid) nucleosides; phosphorothioateinternucleotide linkages; abasic moieties; or a terminal cap moiety atthe 3′-end, 5′-end, or both 3′ and 5′ ends of the oligonucleotide.Non-limiting examples of chemically modified siRNA or siNA molecules aredescribed in Table III herein and in Beigelman et al., U.S. Ser. No.10/444,853, incorporated by reference herein in its entirety includingthe drawings.

By “column format” is meant, solid phase synthesis wherein the solidsupport (for example, CPG, polystyrene) is loaded into a retainingdevice comprising a column, cartridge, or equivalent, which allows thesolid support to be sequentially exposed to reagents suitable for thesynthesis of polymeric molecules, for example, oligonucleotides andtheir derivatives.

In an additional preferred embodiment, the process for deprotection ofmolecules comprising one or more ribonucleotides of the presentinvention is used to deprotect a molecule synthesized using a multi-wellplate format. Specifically, the instant invention provides a highthroughput deprotection of oligonucleotides in a multi-well plate format(for example, a 96-well plate or a 256 well plate). More specificallyrapid deprotection of enzymatic nucleic acid molecules in greater thanmicrogram quantities with high biological activity is featured. It hasbeen determined that the recovery of enzymatically active nucleic acidmolecules in high yield and quantity is dependent upon certain criticalsteps used during its deprotection.

In additional embodiments, the process for deprotection of moleculescomprising one or more ribonucleotides of the present invention is usedto deprotect a molecule synthesized in both a trityl-on and trityl-offmanner.

By “trityl-on” is meant, a molecule, for example an oligonucleotide,synthesized in a manner which leaves the 5′-terminal dimethoxytritylprotecting group or an equivalent protecting group intact.

By “trityl-off” is meant, a molecule, for example an oligonucleotide,synthesized in a manner which removes the 5′-terminal dimethoxytritylprotecting group or an equivalent protecting group.

By “solid phase” is meant, synthesis comprising a solid support (forexample, polystyrene or controlled pore glass) which is used as ascaffold for the sequential addition of subunits in the synthesis of apolymeric molecule such as an oligonucleotide. The solid support can beexposed sequentially to reagents in solution, thereby eliminating theneed for repeated purification and isolation steps during synthesis. Alinker molecule can be used as an interface between the solid supportand the growing polymer. Solid phase synthesis can be used for bothphosphoramidite and H-phosphonate methods of oligonucleotide synthesis.

By “solution phase” is meant, synthesis comprising the combining ofreactants and reagents in solution, such as in a solvent which providesa homogenious mixture. Solution phase synthesis can be a preferredmethod for the synthesis of molecules in large quantities inconsideration of lower costs, more efficient reactivity of reagents, andengineering factors.

By “hybrid phase” is meant, synthesis comprising both solid phase andsolution phase synthesis elements.

The instant invention also features a large scale deprotection method ofmolecules comprising one or more ribonucleotides (for example, 3 mmolsynthesis scale or greater). More specifically rapid deprotection ofmolecules comprising one or more ribonucleotides in greater thanmultigram or kilogram quantities with high biological activity isfeatured. It will be recognized by those skilled in the art thatmodifications concerning time and temperature parameters can be used tooptimize deprotection conditions for reactions of differing scale and/ormolecules of differing composition. The use of different time andtemperature parameters for varying molecular content and/or differentreaction scale applications is hence within the scope of the invention.

In a preferred embodiment, the invention features a method for thepurification of nucleic acid molecules of the instant invention.Specifically, the invention features the use of ethanol or acetonitrile,with ethanol preferred, as an organic modifier in the purification ofoligonucleotides with anion exchange chromatography. In an additionalaspect, the instant invention features the use of ethanol as an organicmodifier used in the purification of oligonucleotide molecules,including but not limited to short interfering RNA (siRNA), enzymaticnucleic acids, antisense nucleic acids, and/or aptamers. In anotherembodiment, the use of an organic modifier is not employed during largescale purification to avoid the use of organic solvents and a solutionof about 20 mM sodium phosphate and about 0.1 M NaCl is used instead.

In additional embodiments, the media used for the purification ofnucleic acid molecules of the instant invention comprises PharmaciaSource Q15 and Biorad Macroprep 25Q type media, or the equivalentthereof such as Pharmacia Q-sepharose, Perceptive POROS HQ, TOSOHAASQ-5PW-HR, Q-5PW, or super Q-5PW. In another embodiment, the purificationmedia is equilibrated with a buffer comprising about 20 mM sodiumphosphate and about 0.1 M NaCl. In yet another embodiment, thepurification media is equilibrated with a buffer comprising either 20%ethanol or acetonitrile in about 20 mM sodium phosphate and about 0.1 MNaCl.

In one embodiment, the invention features a loading buffer foroligonucleotide purification comprising about 20 mM sodium phosphate andabout 0.1 M NaCl. In another embodiment, the invention features aloading buffer for oligonucleotide purification comprising either 20%ethanol or acetonitrile in about 20 mM sodium phosphate and about 0.1 MNaCl. In one aspect, the invention concerns applying a suitable gradientof about 1.0 M NaCl as an elution buffer for the purification of nucleicacid molecules of the instant invention. In another embodiment, theinvention features the analysis of the fractions resulting from thepurification process described herein, by a suitable technique (forexample, UV, HPLC, and/or CGE), and allowing for the pure fractions tobe pooled and desalted via tangential flow filtration or the equivalentthereof, by using membranes comprising such membranes as those selectedfrom the group consisting of Sartorius or Pall Filtron PES 1 Kmembranes, In yet another preferred embodiment, the invention featuresthe use of lyophilization as a means to concentrate the purifiedmaterial.

In one embodiment, a method of purification of siRNA oligonucleotides ofthe invention further comprises the step of hybridizing two purifiedoligonucleotide strands together to form a siRNA duplex.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a β-D-ribo-furanose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA. The RNA can be a siRNA, an enzymatic nucleicacid, antisense nucleic acid, decoy RNA, aptamer RNA, triplex formingoligonucleotide, chimeric RNA, 2-5A antisense chimera, agonist RNA,antagonist RNA, or any other RNA species. RNA can be used for purposesincluding but not limited to use as therapeutic agents, diagnosticreagents, and research reagents.

By “nucleic acid”, “nucleic acid molecule” or “oligonucleotide” as usedherein is meant a molecule having two or more nucleotides. The nucleicacid can be single, double, or multiple stranded and may comprisemodified or unmodified nucleotides or non-nucleotides or variousmixtures and combinations thereof.

In one embodiment, a process of the invention is used for the synthesis,deprotection, and purification of an enzymatic nucleic acid molecule,preferably in the hammerhead, AH ribozyme, NCH (Inozyme), G-cleaver,amberzyme, and/or zinzyme motif.

In one embodiment, a process of the invention is used for the synthesis,deprotection, and purification of a siRNA molecule.

In one embodiment, the synthesis of a double-stranded siRNA molecule ofthe invention, which can include one or more chemical modifications,comprises: (a) synthesis of two complementary strands of the siRNAmolecule; (b) annealing the two complementary strands together underconditions suitable to obtain a double-stranded siRNA molecule. Inanother embodiment, synthesis of the two complementary strands of thesiRNA molecule is by solid phase oligonucleotide synthesis. In yetanother embodiment, synthesis of the two complementary strands of thesiRNA molecule is by solid phase tandem oligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing adouble-stranded siRNA duplex molecule comprising: (a) synthesizing afirst oligonucleotide sequence strand of the siRNA molecule, wherein thefirst oligonucleotide sequence strand comprises a cleavable linkermolecule that can be used as a scaffold for the synthesis of the secondoligonucleotide sequence strand of the siRNA; (b) synthesizing thesecond oligonucleotide sequence strand of siRNA on the scaffold of thefirst oligonucleotide sequence strand, wherein the secondoligonucleotide sequence strand further comprises a chemical moiety thancan be used to purify the siRNA duplex; (c) cleaving the linker moleculeof (a) under conditions suitable for the two siRNA oligonucleotidestrands to hybridize and form a stable duplex; and (d) purifying thesiRNA duplex utilizing the chemical moiety of the second oligonucleotidesequence strand. In one embodiment, cleavage of the linker molecule in(c) above takes place during deprotection of the oligonucleotide, forexample under hydrolysis conditions using an alkylamine base such asmethylamine. In one embodiment, the method of synthesis comprises solidphase synthesis on a solid support such as controlled pore glass (CPG)or polystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity as thesolid support derivatized linker, such that cleavage of the solidsupport derivatized linker and the cleavable linker of (a) takes placeconcomitantly. In another embodiment, the chemical moiety of (b) thatcan be used to isolate the attached oligonucleotide sequence comprises atrityl group, for example a dimethoxytrityl group, which can be employedin a trityl-on synthesis strategy as described herein. In yet anotherembodiment, the chemical moiety, such as a dimethoxytrityl group, isremoved during purification, for example, using acidic conditions.

In a further embodiment, the method for double-stranded siRNA synthesisis a solution phase synthesis or hybrid phase synthesis wherein bothstrands of the siRNA duplex are synthesized in tandem using a cleavablelinker attached to the first sequence which acts a scaffold forsynthesis of the second sequence. Cleavage of the linker underconditions suitable for hybridization of the separate siRNA sequencestrands results in formation of the double-stranded siRNA molecule.

In another embodiment, the invention features a method for synthesizinga double-stranded siRNA duplex molecule comprising: (a) synthesizing oneoligonucleotide sequence strand of the siRNA molecule, wherein thesequence comprises a cleavable linker molecule that can be used as ascaffold for the synthesis of another oligonucleotide sequence; (b)synthesizing a second oligonucleotide sequence having complementarity tothe first sequence strand on the scaffold of (a), wherein the secondsequence comprises the other strand of the double-stranded siRNAmolecule and wherein the second sequence further comprises a chemicalmoiety than can be used to isolate the attached oligonucleotidesequence; (c) purifying the product of (b) utilizing the chemical moietyof the second oligonucleotide sequence strand under conditions suitablefor isolating the full-length sequence comprising both siRNAoligonucleotide strands connected by the cleavable linker and underconditions suitable for the two siRNA oligonucleotide strands tohybridize and form a stable duplex. In one embodiment, cleavage of thelinker molecule in (c) above takes place during deprotection of theoligonucleotide, for example under hydrolysis conditions. In anotherembodiment, cleavage of the linker molecule in (c) above takes placeafter deprotection of the oligonucleotide. In another embodiment, themethod of synthesis comprises solid phase synthesis on a solid supportsuch as controlled pore glass (CPG) or polystyrene, wherein the firstsequence of (a) is synthesized on a cleavable linker, such as a succinyllinker, using the solid support as a scaffold. The cleavable linker in(a) used as a scaffold for synthesizing the second strand can comprisesimilar reactivity or differing reactivity as the solid supportderivatized linker, such that cleavage of the solid support derivatizedlinker and the cleavable linker of (a) takes place either concomitantlyor sequentially. In one embodiment, the chemical moiety of (b) that canbe used to isolate the attached oligonucleotide sequence comprises atrityl group, for example a dimethoxytrityl group.

In another embodiment, the invention features a method for making adouble-stranded siRNA molecule in a single synthetic process comprising:(a) synthesizing an oligonucleotide having a first and a secondsequence, wherein the first sequence is complementary to the secondsequence, and the first oligonucleotide sequence is linked to the secondsequence via a cleavable linker, and wherein a terminal 5′-protectinggroup, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains onthe oligonucleotide having the second sequence; (b) deprotecting theoligonucleotide whereby the deprotection results in the cleavage of thelinker joining the two oligonucleotide sequences; and (c) purifying theproduct of (b) under conditions suitable for isolating thedouble-stranded siRNA molecule, for example using a trityl-on synthesisstrategy as described herein.

The term “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically-modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication, for example by mediating RNA interference “RNAi”or gene silencing in a sequence-specific manner; see for example Bass,2001, Nature, 411, 428-429; Elbashir et al., 2001, Nature, 411, 494-498;and Kreutzer et al., International PCT Publication No. WO 00/44895;Zernicka-Goetz et al., International PCT Publication No. WO 01/36646;Fire, International PCT Publication No. WO 99/32619; Plaetinck et al.,International PCT Publication No. WO 00/01846; Mello and Fire,International PCT Publication No. WO 01/29058; Deschamps-Depaillette,International PCT Publication No. WO 99/07409; and Li et al.,International PCT Publication No. WO 00/44914; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237; Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus etal., 2002, RNA, 8, 842-850; Reinhart et al., 2002, Gene & Dev., 16,1616-1626; and Reinhart & Bartel, 2002, Science, 297, 1831). Nonlimiting examples of siNA molecules of the invention are described inBeigelman et al., U.S. Ser. No. 10/444,853, incorporated by referenceherein in its entirety including the drawings. For example the siNA canbe a double-stranded polynucleotide molecule comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof and thesense region having nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof. The siNA can be assembledfrom two separate oligonucleotides, where one strand is the sense strandand the other is the antisense strand, wherein the antisense and sensestrands are self-complementary (i.e. each strand comprises nucleotidesequence that is complementary to nucleotide sequence in the otherstrand; such as where the antisense strand and sense strand form aduplex or double stranded structure, for example wherein the doublestranded region is about 19 base pairs); the antisense strand comprisesnucleotide sequence that is complementary to nucleotide sequence in atarget nucleic acid molecule or a portion thereof and the sense strandcomprises nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. Alternatively, the siNA is assembled froma single oligonucleotide, where the self-complementary sense andantisense regions of the siNA are linked by means of a nucleic acidbased or non-nucleic acid-based linker(s). The siNA can be apolynucleotide with a duplex, asymmetric duplex, hairpin or asymmetrichairpin secondary structure, having self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a separatetarget nucleic acid molecule or a portion thereof and the sense regionhaving nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. The siNA can be a circularsingle-stranded polynucleotide having two or more loop structures and astem comprising self-complementary sense and antisense regions, whereinthe antisense region comprises nucleotide sequence that is complementaryto nucleotide sequence in a target nucleic acid molecule or a portionthereof and the sense region having nucleotide sequence corresponding tothe target nucleic acid sequence or a portion thereof, and wherein thecircular polynucleotide can be processed either in vivo or in vitro togenerate an active siNA molecule capable of mediating RNAi. The siNA canalso comprise a single stranded polynucleotide having nucleotidesequence complementary to nucleotide sequence in a target nucleic acidmolecule or a portion thereof (for example, where such siNA moleculedoes not require the presence within the siNA molecule of nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof), wherein the single stranded polynucleotide can furthercomprise a terminal phosphate group, such as a 5′-phosphate (see forexample Martinez et al., 2002, Cell., 110, 563-574 and Schwarz et al.,2002, Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certainembodiment, the siNA molecule of the invention comprises separate senseand antisense sequences or regions, wherein the sense and antisenseregions are covalently linked by nucleotide or non-nucleotide linkersmolecules as is known in the art, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der waalsinteractions, hydrophobic interactions, and/or stacking interactions. Incertain embodiments, the siNA molecules of the invention comprisenucleotide sequence that is complementary to nucleotide sequence of atarget gene. In another embodiment, the siNA molecule of the inventioninteracts with nucleotide sequence of a target gene in a manner thatcauses inhibition of expression of the target gene. As used herein, siNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically-modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering nucleicacid molecules of the invention lack 2′-hydroxy (2′-OH) containingnucleotides. Applicant describes in certain embodiments shortinterfering nucleic acids that do not require the presence ofnucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of the invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′-OHgroup). Such siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can howeverhave an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides atabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions. Themodified short interfering nucleic acid molecules of the invention canalso be referred to as short interfering modified oligonucleotides“siMON.” As used herein, the term siNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpinRNA (shRNA), short interfering oligonucleotide, short interferingnucleic acid, short interfering modified oligonucleotide,chemically-modified siRNA, post-transcriptional gene silencing RNA(ptgsRNA), and others. In addition, as used herein, the term RNAi ismeant to be equivalent to other terms used to describe sequence specificRNA interference, such as post transcriptional gene silencing,translational inhibition, or epigenetics. For example, siNA molecules ofthe invention can be used to epigenetically silence genes at both thepost-transcriptional level or the pre-transcriptional level. In anon-limiting example, epigenetic regulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure to alter gene expression (see, for example,Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237).

By “asymmetric hairpin” as used herein is meant a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complimentary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin siNA molecule of the invention can comprise an antisense regionhaving length sufficient to mediate RNAi in a cell or in vitro system(e.g. about 19 to about 22 (e.g., about 19, 20, 21, or 22 nucleotides)and a loop region comprising about 4 to about 8 nucleotides, and a senseregion having about 3 to about 18 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, or 18) nucleotides that are complementary tothe antisense region. The asymmetric hairpin siNA molecule can alsocomprise a 5′-terminal phosphate group that can be chemically modified.The loop portion of the asymmetric hairpin siNA molecule can comprisenucleotides, non-nucleotides, linker molecules, or conjugate moleculesas described herein.

By “asymmetric duplex” as used herein is meant a siNA molecule havingtwo separate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complimentarynucleotides to base pair with the antisense region and form a duplex.For example, an asymmetric duplex siNA molecule of the invention cancomprise an antisense region having length sufficient to mediate RNAi ina cell or in vitro system (e.g. about 19 to about 22 nucleotides) and asense region having about 3 to about 18 nucleotides that arecomplementary to the antisense region.

By “enzymatic nucleic acid molecule” it is meant a nucleic acid moleculethat has complementarity in a substrate binding region to a specifiedgene target, and also has an enzymatic activity which is active tospecifically cleave target RNA. That is, the enzymatic nucleic acidmolecule is able to intermolecularly cleave RNA and thereby inactivate atarget RNA molecule. These complementary regions allow sufficienthybridization of the enzymatic nucleic acid molecule to the target RNAand thus permit cleavage. One hundred percent complementarity ispreferred, but complementarity as low as 50-75% may also be useful inthis invention. The nucleic acids may be modified at the base, sugar,and/or phosphate groups. The term enzymatic nucleic acid is usedinterchangeably with phrases such as ribozymes, catalytic RNA, enzymaticRNA, catalytic DNA, aptazyme or aptamer-binding ribozyme, regulatableribozyme, catalytic oligonucleotides, nucleozyme, DNAzyme, RNA enzyme,endoribonuclease, endonuclease, minizyme, leadzyme, oligozyme or DNAenzyme. All of these terminologies describe nucleic acid molecules withenzymatic activity. The specific enzymatic nucleic acid moleculesdescribed in the instant application are not meant to be limiting andthose skilled in the art will recognize that all that is important in anenzymatic nucleic acid molecule of this invention is that it have aspecific substrate binding site which is complementary to one or more ofthe target nucleic acid regions, and that it have nucleotide sequenceswithin or surrounding that substrate binding site which impart a nucleicacid cleaving activity to the molecule (Cech et al., U.S. Pat. No.4,987,071; Cech et al., 1988, JAMA).

By “antisense nucleic acid” it is meant a non-enzymatic nucleic acidmolecule that binds to target RNA by means of RNA-RNA or RNA-DNA orRNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566)interactions and alters the activity of the target RNA (for a review seeStein and Cheng, 1993 Science 261, 1004). Typically, antisense moleculeswill be complementary to a target sequence along a single contiguoussequence of the antisense molecule. However, in certain embodiments, anantisense molecule may bind to substrate such that the substratemolecule forms a loop, and/or an antisense molecule may bind such thatthe antisense molecule forms a loop. Thus, the antisense molecule may becomplementary to two (or even more) non-contiguous substrate sequencesor two (or even more) non-contiguous sequence portions of an antisensemolecule may be complementary to a target sequence or both.

By “AH ribozyme” motif is meant, an enzymatic nucleic acid moleculecomprising a motif as described in Kore et al., 1998, Nucleic AcidsResearch, 26(18), 4116-4120.

By “NCH” or “Inozyme” motif is meant, an enzymatic nucleic acid moleculecomprising a motif as described in Ludwig et al., U.S. Ser. No.09/406,643, filed Sep. 27, 1999, entitled “COMPOSITIONS HAVING RNACLEAVING ACTIVITY”, and International PCT publication Nos. WO 98/58058and WO 98/58057, all incorporated by reference herein in their entiretyincluding the drawings.

By “G-cleaver” motif is meant, an enzymatic nucleic acid moleculecomprising a motif as described in Eckstein et al., International PCTpublication No. WO 99/16871, incorporated by reference herein in itsentirety including the drawings.

By “zinzyme” motif is meant, a class II enzymatic nucleic acid moleculecomprising a motif as described in Beigelman et al., International PCTpublication No. WO 99/55857, incorporated by reference herein in itsentirety including the drawings. Zinzymes represent a non-limitingexample of an enzymatic nucleic acid molecule that does not require aribonucleotide (2′-OH) group within its own nucleic acid sequence foractivity.

By “amberzyme” motif is meant, a class I enzymatic nucleic acid moleculecomprising a motif as described in Beigelman et al., International PCTpublication No. WO 99/55857, incorporated by reference herein in itsentirety including the drawings. Amberzymes represent a non-limitingexample of an enzymatic nucleic acid molecule that does not require aribonucleotide (2′-OH) group within its own nucleic acid sequence foractivity.

By “2-5A antisense chimera” it is meant, an antisense oligonucleotidecontaining a 5′ phosphorylated 2′-5′-linked adenylate residues. Thesechimeras bind to target RNA in a sequence-specific manner and activate acellular 2-5A-dependent ribonuclease which, in turn, cleaves the targetRNA (Torrence et al., 1993 Proc. Natl. Acad. Sci. USA 90, 1300).

By “triplex forming oligonucleotide” it is meant an oligonucleotide thatcan bind to a double-stranded DNA in a sequence-specific manner to forma triple-strand helix. Formation of such triple helix structure has beenshown to inhibit transcription of the targeted gene (Duval-Valentin etal., 1992 Proc. Natl. Acad. Sci. USA 89, 504).

By “decoy RNA” is meant an RNA molecule that mimics the natural bindingdomain for a ligand. The decoy RNA therefore competes with naturalbinding target for the binding of a specific ligand. For example, it hasbeen shown that over-expression of HIV trans-activation response (TAR)RNA can act as a “decoy” and efficiently binds HIV tat protein, therebypreventing it from binding to TAR sequences encoded in the HIV RNA(Sullenger et al., 1990, Cell, 63, 601-608). This is meant to be aspecific example. Those in the art will recognize that this is but oneexample, and other embodiments can be readily generated using techniquesgenerally known in the art.

By “agonist RNA” is meant an RNA molecule that can bind to proteinreceptors with high affinity and cause the stimulation of specificcellular pathways.

By “antagonist RNA” is meant an RNA molecule that can bind to cellularproteins and prevent it from performing its normal biological function(for example, see Tsai et al., 1992 Proc. Natl. Acad. Sci. USA 89,8864-8868).

By “aptamer” or “nucleic acid aptamer” as used herein is meant a nucleicacid molecule that binds specifically to a target molecule wherein thenucleic acid molecule has sequence that comprises a sequence recognizedby the target molecule in its natural setting. Alternately, an aptamercan be a nucleic acid molecule that binds to a target molecule where thetarget molecule does not naturally bind to a nucleic acid. The targetmolecule can be any molecule of interest. For example, the aptamer canbe used to bind to a ligand-binding domain of a protein, therebypreventing interaction of the naturally occurring ligand with theprotein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art. (See, for example, Gold et al.,1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J.Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser,2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287,820; and Jayasena, 1999, Clinical Chemistry, 45, 1628).

By “comprising” is meant including, but not limited to, whatever followsthe word “comprising”. Thus, use of the term “comprising” indicates thatthe listed elements are required or mandatory, but that other elementsare optional and may or may not be present. By “consisting of” is meantincluding, and limited to, whatever follows the phrase “consisting of”.Thus, the phrase “consisting of” indicates that the listed elements arerequired or mandatory, and that no other elements may be present. By“consisting essentially of” is meant including any elements listed afterthe phrase, and limited to other elements that do not interfere with orcontribute to the activity or action specified in the disclosure for thelisted elements. Thus, the phrase “consisting essentially of” indicatesthat the listed elements are required or mandatory, but that otherelements are optional and may or may not be present depending uponwhether or not they affect the activity or action of the listedelements.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings will first briefly be described.

Drawings:

FIG. 1 is aschematic representation of a one pot deprotection of nucleicacid molecules comprising one or more ribonucleotides synthesized usingthe phosphoramidite approach.

FIG. 2 is schematic representation of incomplete N-phthaloyldeprotection products. Compound A represents intact N-phthaloylprotection, compound B represents partially cleaved N-phthaloylprotection, and compound C represents a free 2′-amino group aftercomplete cleavage of N-phthaloyl protection.

FIG. 3 shows a comparison of different one pot deprotection methodsbased on electrospray mass spectrometry (ESMS) data. FIG. 3A shows aESMS chromatogram of a purified full length oligonucleotide containingribonucleotide functions (TBDMS protection) and two 2′-amino functions(N-phthaloyl protection) following a deprotection method which utilizedanhydrous methylamine/DMSO/TEA.3HF. FIG. 3B shows a ESMS chromatogram ofa purified full length oligonucleotide containing ribonucleotidefunctions (TBDMS protection) and two 2′-amino functions (N-phthaloylprotection) following a deprotection method which utilized aqueousmethylamine/DMSO/TEA.3HF. The three peaks seen in FIG. 3A represents themasses of the fully deprotected oligo, the deprotected oligo with onepartially deprotected phthaloyl group intact, and the deprotected oligowith two partially deprotected phthaloyl groups intact. The single peakshown in FIG. 3B represents the mass of the fully deprotected oligoonly.

FIG. 4 shows a comparison of different one pot deprotection methodsbased on capillary gel electrophoresis data. FIG. 4A shows a CEchromatogram of the purified full length oligonucleotide shown in FIG.3A, which results in a broad peak due to partially cleaved phthaloylgroup contaminants. FIG. 4B shows a CE chromatogram of the purified fulllength oligonucleotide shown in FIG. 3B, which results in a singlenarrow peak consistent with a homogenous oligonucleotide species.

FIG. 5 shows a non-limiting example of a scheme for the synthesis ofdouble-stranded siNA molecules. The complementary siNA sequence strands,strand 1 and strand 2, are synthesized in tandem and are connected by acleavable linkage, such as a nucleotide succinate or abasic succinate,which can be the same or different from the cleavable linker used forsolid phase synthesis on a solid support. The synthesis can be eithersolid phase or solution phase, in the example shown, the synthesis is asolid phase synthesis. The synthesis is performed such that a protectinggroup, such as a dimethoxytrityl group, remains intact on the terminalnucleotide of the tandem oligonucleotide. Upon cleavage and deprotectionof the oligonucleotide, the two siNA strands spontaneously hybridize toform a siNA duplex, which allows the purification of the duplex byutilizing the properties of the terminal protecting group, for exampleby applying a trityl on purification method wherein onlyduplexes/oligonucleotides with the terminal protecting group areisolated.

FIG. 6 shows a MALDI-TOF mass spectrum of a purified double-strandedsiNA duplex synthesized by a method of the invention. The two peaksshown correspond to the predicted mass of the separate siNA sequencestrands. This result demonstrates that the siNA duplex generated fromtandem synthesis can be purified as a single entity using a simpletrityl-on purification methodology.

FIG. 7 shows non-limiting examples of different stabilizationchemistries (1-10) that can be used, for example, to stabilize the3′-end of siNA sequences of the invention, including (1) [3-3′]-inverteddeoxyribose; (2) deoxyribonucleotide; (3)[5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5)[5′-3′]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7)[3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9)[5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotide. Inaddition to modified and unmodified backbone chemistries indicated inthe figure, these chemistries can be combined with different backbonemodifications as described herein, for example, backbone modificationshaving Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to theterminal modifications shown can be another modified or unmodifiednucleotide or non-nucleotide.

DETAILED DESCRIPTION OF THE INVENTION

Nucleic Acid Molecule Mediating RNA Interfernece (RNAi):

RNA interference refers to the process of sequence specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes whichis commonly shared by diverse flora and phyla (Fire et al., 1999, TrendsGenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or the random integration of transposonelements into a host genome via a cellular response that specificallydestroys homologous single-stranded RNA or viral genomic RNA. Thepresence of dsRNA in cells triggers the RNAi response though a mechanismthat has yet to be fully characterized. This mechanism appears to bedifferent from the interferon response that results from dsRNA-mediatedactivation of protein kinase PKR and 2′, 5′-oligoadenylate synthetaseresulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from Dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes. Dicer has also been implicated in the excision of 21- and22-nucleotide small temporal RNAs (stRNAs) from precursor RNA ofconserved structure that are implicated in translational control(Hutvagner et al., 2001, Science, 293, 834). The RNAi response alsofeatures an endonuclease complex containing a siRNA, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single-stranded RNA having sequence homologous to the siRNA. Cleavageof the target RNA takes place in the middle of the region complementaryto the guide sequence of the siRNA duplex (Elbashir et al., 2001, GenesDev., 15, 188). In addition, RNA interference can also involve small RNA(e.g., micro-RNA or miRNA) mediated gene silencing, presumably thoughcellular mechanisms that regulate chromatin structure and therebyprevent transcription of target gene sequences (see for exampleAllshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237). As such, siNA molecules of theinvention can be used to mediate gene silencing via interaction with RNAtranscripts or alternately by interaction with particular genesequences, wherein such interaction results in gene silencing either atthe transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans. Wiannyand Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated bydsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describeRNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexes ofsynthetic 21-nucleotide RNAs in cultured mammalian cells including humanembryonic kidney and HeLa cells. Recent work in Drosophila embryoniclysates has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21 nucleotidesiRNA duplexes are most active when containing two 2-nucleotide3′-terminal nucleotide overhangs. Furthermore, substitution of one orboth siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishesRNAi activity, whereas substitution of 3′-terminal siRNA nucleotideswith deoxy nucleotides was shown to be tolerated. Mismatch sequences inthe center of the siRNA duplex were also shown to abolish RNAi activity.In addition, these studies also indicate that the position of thecleavage site in the target RNA is defined by the 5′-end of the siRNAguide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J.,20, 6877). Other studies have indicated that a 5′-phosphate on thetarget-complementary strand of a siRNA duplex is required for siRNAactivity and that ATP is utilized to maintain the 5′-phosphate moiety onthe siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNAmolecules lacking a 5′-phosphate are active when introduced exogenously,suggesting that 5′-phosphorylation of siRNA constructs may occur invivo. The term short interfering RNA (siRNA) is used interchangeablywith the terms short interfering nucleic acid (siNA), double-strandedRNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA (shRNA), all ofwhich are molecules capable of mediating RNA interference (RNAi)

Enzymatic Nucleic Acid Molecules:

The enzymatic RNA molecule is a nucleic acid molecule comprising one ormore ribonucleotides. Enzymatic RNA molecule is able to intramolecularlyor intermolecularly cleave RNA or DNA and thereby inactivate a targetRNA or DNA molecule. The enzymatic RNA acid molecule that hascomplementarity in a substrate binding region to a specified genetarget, also has an enzymatic activity that specifically cleaves RNA orDNA in that target. This complementarity functions to allow sufficienthybridization of the enzymatic RNA molecule to the target RNA or DNA toallow the cleavage to occur. 100% Complementarity is preferred, butcomplementarity as low as 50-75% may also be useful in this invention.The nucleic acids may be modified at the base, sugar, and/or phosphategroups.

The term enzymatic RNA acid is used interchangeably with phrases such asribozymes, enzymatic nucleic acid, catalytic RNA, enzymatic RNA,nucleozyme, RNA enzyme, endoribonuclease, minizyme, leadzyme, oligozymeand the like.

By “complementarity” is meant a nucleic acid that can form hydrogenbond(s) with other RNA sequence by either traditional Watson-Crick orother non-traditional types (for example, Hoogsteen type) of base-pairedinteractions.

RNA molecules having an endonuclease enzymatic activity are able torepeatedly cleave other separate RNA molecules in a nucleotide basesequence-specific manner. Such enzymatic RNA molecules can be targetedto virtually any RNA transcript, and efficient cleavage achieved invitro (Zaug et al., 324, Nature 429 1986; Kim et al., 84 Proc. Natl.Acad. Sci. USA 8788, 1987; Haseloff and Gerlach, 334 Nature 585, 1988;Cech, 260 JAMA 3030, 1988; and Jefferies et al., 17 Nucleic AcidsResearch 1371, 1989).

Because of their sequence-specificity, trans-cleaving ribozymes showpromise as therapeutic agents for human disease (Usman & McSwiggen, 1995Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med.Chem. 38, 2023-2037). Ribozymes can be designed to cleave specific RNAtargets within the background of cellular RNA. Such a cleavage eventrenders the mRNA non-functional and abrogates protein expression fromthat RNA. In this manner, synthesis of a protein associated with adisease state can be selectively inhibited.

Seven basic varieties of naturally-occurring enzymatic RNAs are knownpresently. Each can catalyze the hydrolysis of RNA phosphodiester bondsin trans (and thus can cleave other RNA molecules) under physiologicalconditions. In general, enzymatic RNA act by first binding to a targetRNA. Such binding occurs through the target binding portion of aenzymatic nucleic acid which is held in close proximity to an enzymaticportion of the molecule that acts to cleave the target RNA. Thus, theenzymatic nucleic acid first recognizes and then binds a target RNAthrough complementary base-pairing, and once bound to the correct site,acts enzymatically to cut the target RNA. Strategic cleavage of such atarget RNA will destroy its ability to direct synthesis of an encodedprotein. After an enzymatic nucleic acid has bound and cleaved its RNAtarget, it is released from that RNA to search for another target andcan repeatedly bind and cleave new targets. In addition, several invitro selection (evolution) strategies (Orgel, 1979, Proc. R. Soc.London, B 205, 435) have been used to evolve new nucleic acid catalystscapable of catalyzing cleavage and ligation of phosphodiester linkages(Joyce, 1989, Gene, 82, 83-87; Beaudry et al., 1992, Science 257,635-641; Joyce, 1992, Scientific American 267, 90-97; Breaker et al.,1994, TIBTECH 12, 268; Bartel et al., 1993, Science 261:1411-1418;Szostak, 1993, TIBS 17, 89-93; Kumar et al., 1995, FASEB J., 9, 1183;Breaker, 1996, Curr. Op. Biotech., 7, 442; Santoro et al., 1997, Proc.Natl. Acad. Sci., 94, 4262; Tang et al., 1997, RNA 3, 914; Nakamaye &Eckstein, 1994, supra; Long & Uhlenbeck, 1994, supra; Ishizaka et al.,1995, supra; Vaish et al., 1997, Biochemistry 36, 6495; all of these areincorporated by reference herein). Each can catalyze a series ofreactions including the hydrolysis of phosphodiester bonds in trans (andthus can cleave other RNA molecules) under physiological conditions.

The enzymatic nature of a ribozyme has significant advantages, such asthe concentration of ribozyme necessary to affect a therapeutictreatment is lower. This advantage reflects the ability of the ribozymeto act enzymatically. Thus, a single ribozyme molecule is able to cleavemany molecules of target RNA. In addition, the ribozyme is a highlyspecific inhibitor, with the specificity of inhibition depending notonly on the base-pairing mechanism of binding to the target RNA, butalso on the mechanism of target RNA cleavage. Single mismatches, orbase-substitutions, near the site of cleavage can be chosen tocompletely eliminate catalytic activity of a ribozyme.

Nucleic acid molecules having an endonuclease enzymatic activity areable to repeatedly cleave other separate RNA molecules in a nucleotidebase sequence-specific manner. Such enzymatic nucleic acid molecules canbe targeted to virtually any RNA transcript, and achieve efficientcleavage in vitro (Zaug et al., 324, Nature 429 1986; Uhlenbeck, 1987Nature 328, 596; Kim et al., 84 Proc. Natl. Acad. Sci. USA 8788, 1987;Dreyfus, 1988, Einstein Quart. J. Bio. Med., 6, 92; Haseloff andGerlach, 334 Nature 585, 1988; Cech, 260 JAMA 3030, 1988; and Jefferieset al., 17 Nucleic Acids Research 1371, 1989; Santoro et al., 1997supra).

Because of their sequence specificity, trans-cleaving ribozymes showpromise as therapeutic agents for human disease (Usman & McSwiggen, 1995Ann. Rep. Med. Chem. 30, 285-294; Christoffersen and Marr, 1995 J. Med.Chem. 38, 2023-2037). Ribozymes can be designed to cleave specific RNAtargets within the background of cellular RNA. Such a cleavage eventrenders the RNA non-functional and abrogates protein expression fromthat RNA. In this manner, synthesis of a protein associated with adisease state can be selectively inhibited (Warashina et al., 1999,Chemistry and Biology, 6, 237-250).

In one aspect, enzymatic nucleic acid molecules are formed in ahammerhead or hairpin motif, but may also be formed in the motif of ahepatitis delta virus (HDV), group I intron, RNaseP RNA (in associationwith an external guide sequence) or Neurospora VS RNA. Examples of suchhammerhead motifs are described by Rossi et al., 1992, Aids Research andHuman Retroviruses 8, 183; Usman et al., 1996, Curr. Op. Struct. Biol.,1, 527; of hairpin motifs by Hampel et al., EP 0360257; Hampel andTritz, 1989 Biochemistry 28, 4929; and Hampel et al., 1990 Nucleic AcidsRes. 18, 299; Chowrira et al., U.S. Pat. No. 5,631,359; an example ofthe hepatitis delta virus motif is described by Perrotta and Been, 1992Biochemistry 31, 16; Been et al., U.S. Pat. No. 5,625,047; of the RNasePmotif by Guerrier-Takada et al., 1983 Cell 35, 849; Forster and Altman,1990 Science 249, 783; Neurospora VS RNA ribozyme motif is described byCollins (Saville and Collins, 1990 Cell 61, 685-696; Saville andCollins, 1991 Proc. Natl. Acad. Sci. USA 88, 8826-8830; Guo and Collins,1995 EMBO J. 14, 368) and of the Group I intron by Zaug et al., 1986,Nature, 324, 429; Cech et al., U.S. Pat. No. 4,987,071. These specificmotifs are not limiting in the invention and those skilled in the artwill recognize that all that is important in an enzymatic nucleic acidmolecule with endonuclease activity of this invention is that it has aspecific substrate binding site which is complementary to one or more ofthe target gene RNA and that it have nucleotide sequences within orsurrounding that substrate binding site which impart an RNA cleavingactivity to the molecule. The length of the binding site varies fordifferent ribozyme motifs, and a person skilled in the art willrecognize that to achieve an optimal ribozyme activity the length of thebinding arm should be of sufficient length to form a stable interactionwith the target nucleic acid sequence.

Catalytic activity of the ribozymes described in the instant inventioncan be optimized as described by Draper et al., supra. The details willnot be repeated here, but include altering the length of the ribozymebinding arms, or chemically synthesizing ribozymes with modifications(base, sugar and/or phosphate) that prevent their degradation by serumribonucleases and/or enhance their enzymatic activity (see e.g.,Eckstein et al., International Publication No. WO 92/07065; Perrault etal., 1990 Nature 344, 565; Pieken et al., 1991 Science 253, 314; Usmanand Cedergren, 1992 Trends in Biochem. Sci. 17, 334; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; and Burgin et al., supra; all of these describe variouschemical modifications that can be made to the base, phosphate and/orsugar moieties of enzymatic RNA molecules). Modifications which enhancetheir efficacy in cells, and removal of bases from stem loop structuresto shorten RNA synthesis times and reduce chemical requirements aredesired. (All these publications are hereby incorporated by referenceherein).

Aptamers:

Nucleic acid aptamers can be selected to specifically bind to aparticular ligand of interest (see for example Gold et al., U.S. Pat.No. 5,567,588 and U.S. Pat. No. 5,475,096, Gold et al., 1995, Annu. Rev.Biochem., 64, 763; Brody and Gold, 2000, J. Biotechnol., 74, 5; Sun,2000, Curr. Opin. Mol. Ther., 2, 100; Kusser, 2000, J. Biotechnol., 74,27; Hermann and Patel, 2000, Science, 287, 820; and Jayasena, 1999,Clinical Chemistry, 45, 1628). For example, the use of in vitroselection can be applied to evolve nucleic acid aptamers with bindingspecificity for HIV envelope glycoprotein gp41, gp120 or to any otherportion of HIV that disrupts fusogenic activity of the virus. Nucleicacid aptamers can include chemical modifications and linkers asdescribed herein. Nucleic apatmers of the invention can be doublestranded or single stranded and can comprise one distinct nucleic acidsequence or more than one nucleic acid sequences complexed with oneanother. Aptamer molecules of the invention that bind to HIV envelopeglycoprotein, for example gp41, can modulate the fusogenic activity ofHIV and therefore modulate cell entry and infectivity of the virus.

Antisense:

Antisense molecules can be modified or unmodified RNA, DNA, or mixedpolymer oligonucleotides and primarily function by specifically bindingto matching sequences resulting in modulation of peptide synthesis(Wu-Pong, November 1994, BioPharm, 20-33). The antisense oligonucleotidebinds to target RNA by Watson Crick base-pairing and blocks geneexpression by preventing ribosomal translation of the bound sequenceseither by steric blocking or by activating RNase H enzyme. Antisensemolecules may also alter protein synthesis by interfering with RNAprocessing or transport from the nucleus into the cytoplasm(Mukhopadhyay & Roth, 1996, Crit. Rev. in Oncogenesis 7, 151-190).

In addition, binding of single stranded DNA to RNA may result innuclease degradation of the heteroduplex (Wu-Pong, supra; Crooke,supra). To date, the only backbone modified DNA chemistry which will actas substrates for RNase H are phosphorothioates, phosphorodithioates,and borontrifluoridates. Recently, it has been reported that 2′-arabinoand 2′-fluoro arabino-containing oligos can also activate RNase Hactivity.

A number of antisense molecules have been described that utilize novelconfigurations of chemically modified nucleotides, secondary structure,and/or RNase H substrate domains (Woolf et al., U.S. Pat. No. 5,989,912;Thompson et al., U.S. Ser. No. 60/082,404 which was filed on Apr. 20,1998; Hartmann et al., U.S. Ser. No. 60/101,174 which was filed on Sep.21, 1998) all of these are incorporated by reference herein in theirentirety.

Antisense DNA can be used to target RNA by means of DNA-RNAinteractions, thereby activating RNase H, which digests the target RNAin the duplex. Antisense DNA can be chemically synthesized or can beexpressed via the use of a single stranded DNA intracellular expressionvector or the equivalent thereof.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) that prevent their degradation by serumribonucleases can increase their potency (see e.g., Eckstein et al.,International Publication No. WO 92/07065; Perrault et al., 1990 Nature344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren,1992, Trends in Biochem. Sci. 17, 334; Usman et al., InternationalPublication No. WO 93/15187; Rossi et al., International Publication No.WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; and Burgin et al., supra;all of these describe various chemical modifications that can be made tothe base, phosphate and/or sugar moieties of the nucleic acid moleculesdescribed herein. All these references are incorporated by referenceherein. Modifications which enhance their efficacy in cells, and removalof bases from nucleic acid molecules to shorten oligonucleotidesynthesis times and reduce chemical requirements are desired.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-flouro,2′-O-methyl, 2′-H, nucleotide base modifications (for a review see Usmanand Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic AcidsSymp. Ser. 31, 163; Burgin et al., 1996, Biochemistry , 35, 14090).Sugar modifications of nucleic acid molecules have been extensivelydescribed in the art (see Eckstein et al., International Publication PCTNo. WO 92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken etal. Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem.Sci. , 1992, 17, 334-339; Usman et al. International Publication PCT No.WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995,J. Biol. Chem., 270, 25702; Beigelman et al., International PCTpublication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824;Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCTPublication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404which was filed on Apr. 20, 1998; Karpeisky et al., 1998, TetrahedronLett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers (Nucleic acidSciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev. Biochem., 67,99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; allof the references are hereby incorporated by reference herein in theirtotalities). Such publications describe general methods and strategiesto determine the location of incorporation of sugar, base and/orphosphate modifications and the like into ribozymes without inhibitingcatalysis. In view of such teachings, similar modifications can be usedas described herein to modify the nucleic acid molecules of the instantinvention.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorothioate, and/or 5′-methylphosphonatelinkages improves stability, too many of these modifications may causesome toxicity. Therefore when designing nucleic acid molecules theamount of these internucleotide linkages should be minimized. Thereduction in the concentration of these linkages should lower toxicityresulting in increased efficacy and higher specificity of thesemolecules.

Nucleic acid molecules having chemical modifications which maintain orenhance activity are provided. Such nucleic acid is also generally moreresistant to nucleases than unmodified nucleic acid. Thus, in a celland/or in vivo the activity may not be significantly lowered.Therapeutic nucleic acid molecules delivered exogenously must optimallybe stable within cells until translation of the target RNA has beeninhibited long enough to reduce the levels of the undesirable protein.This period of time varies between hours to days depending upon thedisease state. Clearly, nucleic acid molecules must be resistant tonucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of RNA and DNA (Wincottet al., 1995 Nucleic Acids Res. 23, 2677; Caruthers et al., 1992,Methods in Enzymology 211,3-19 (incorporated by reference herein) haveexpanded the ability to modify nucleic acid molecules by introducingnucleotide modifications to enhance their nuclease stability asdescribed above.

Use of these the nucleic acid-based molecules of the invention will leadto better treatment of disease progression by affording the possibilityof combination therapies (e.g., multiple antisense or enzymatic nucleicacid molecules targeted to different genes, nucleic acid moleculescoupled with known small molecule inhibitors, or intermittent treatmentwith combinations of molecules (including different motifs) and/or otherchemical or biological molecules). The treatment of patients withnucleic acid molecules may also include combinations of different typesof nucleic acid molecules.

Therapeutic nucleic acid molecules (e.g., enzymatic nucleic acidmolecules and antisense nucleic acid molecules) delivered exogenouslymust optimally be stable within cells until translation of the targetRNA has been inhibited long enough to reduce the levels of theundesirable protein. This period of time varies between hours to daysdepending upon the disease state. Clearly, these nucleic acid moleculesmust be resistant to nucleases in order to function as effectiveintracellular therapeutic agents. Improvements in the chemical synthesisof nucleic acid molecules described in the instant invention and in theart have expanded the ability to modify nucleic acid molecules byintroducing nucleotide modifications to enhance their nuclease stabilityas described above.

By “enhanced enzymatic activity” is meant to include activity measuredin cells and/or in vivo where the activity is a reflection of bothcatalytic activity and ribozyme stability. In this invention, theproduct of these properties is increased or not significantly (less than10-fold) decreased in vivo compared to an all RNA ribozyme or all DNAenzyme.

In yet another preferred embodiment, nucleic acid catalysts havingchemical modifications which maintain or enhance enzymatic activity areprovided. Such nucleic acid is also generally more resistant tonucleases than unmodified nucleic acid. Thus, in a cell and/or in vivothe activity may not be significantly lowered. As exemplified hereinsuch nucleic acid molecules are useful in a cell and/or in vivo even ifactivity over all is reduced 10 fold (see for example Burgin et al.,1996, Biochemistry, 35, 14090). Such nucleic acids herein are said to“maintain” the activity of an all RNA nucleic acid molecule, such as aribozyme or siRNA.

In another aspect the nucleic acid molecules comprise a 5′ and/or a3′-cap structure.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Wincott et al., WO 97/26270, incorporated by reference herein).These terminal modifications protect the nucleic acid molecule fromexonuclease degradation, and may help in delivery and/or localizationwithin a cell. The cap may be present at the 5′-terminus (5′-cap) or atthe 3′-terminus (3′-cap) or may be present on both termini. Innon-limiting examples the 5′-cap is selected from the group comprisinginverted abasic residue (moiety), 4′,5′-methylene nucleotide;1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide; 1,5-anhydrohexitol nucleotide; L-nucleotides;alpha-nucleotides; modified base nucleotide; phosphorodithioate linkage;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; acyclic3,4-dihydroxybutyl nucleotide; acyclic 3,5-dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety; 3′-3′-inverted abasic moiety;3′-2′-inverted nucleotide moiety; 3′-2′-inverted abasic moiety;1,4-butanediol phosphate; 3′-phosphoramidate; hexylphosphate; aminohexylphosphate; 3′-phosphate; 3′-phosphorothioate; phosphorodithioate; orbridging or non-bridging methylphosphonate moiety (for more details seeWincott et al., International PCT publication No. WO 97/26270,incorporated by reference herein).

In yet another preferred embodiment, the 3′-cap is selected from a groupcomprising, 4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkylphosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate;6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropylphosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide;alpha-nucleotide; modified base nucleotide; phosphorodithioate;threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide;3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide,5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasic moiety;5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediol phosphate;5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details, seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, includingstraight-chain, branched-chain, and cyclic alkyl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably it is a lower alkyl offrom 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group maybe substituted or unsubstituted. When substituted the substitutedgroup(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂,amino, or SH. The term also includes alkenyl groups which areunsaturated hydrocarbon groups containing at least one carbon-carbondouble bond, including straight-chain, branched-chain, and cyclicgroups. Preferably, the alkenyl group has 1 to 12 carbons. Morepreferably it is a lower alkenyl of from 1 to 7 carbons, more preferably1 to 4 carbons. The alkenyl group may be substituted or unsubstituted.When substituted the substituted group(s) is preferably, hydroxyl,cyano, alkoxy, ═O, ═S, NO₂, halogen, N(CH₃)₂, amino, or SH. The term“alkyl” also includes alkynyl groups which have an unsaturatedhydrocarbon group containing at least one carbon-carbon triple bond,including straight-chain, branched-chain, and cyclic groups. Preferably,the alkynyl group has 1 to 12 carbons. More preferably it is a loweralkynyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. Thealkynyl group may be substituted or unsubstituted. When substituted thesubstituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂or N(CH₃)₂, amino or SH.

Such alkyl groups may also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. An “aryl” group refers to anaromatic group which has at least one ring having a conjugated πelectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyano, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above). Carbocyclicaryl groups are groups wherein the ring atoms on the aromatic ring areall carbon atoms. The carbon atoms are optionally substituted.Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo,pyrimidyl, pyrazinyl, imidazolyl and the like, all optionallysubstituted. An “amide” refers to an —C(O)—NH—R, where R is eitheralkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′,where R is either alkyl, aryl, alkylaryl or hydrogen.

By “nucleotide” as used herein is as recognized in the art to includenatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1′ position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, chemically modified nucleotides, modifiednucleotides, non-natural nucleotides, non-standard nucleotides andother; see for example, Usman and McSwiggen, supra; Eckstein et al.,International PCT Publication No. WO 92/07065; Usman et al.,International PCT Publication No. WO 93/15187; Uhlman & Peyman, supraall are hereby incorporated by reference herein). There are severalexamples of modified nucleic acid bases known in the art as summarizedby Limbach et al., 1994, Nucleic Acids Res. 22, 2183. Some of thenon-limiting examples of base modifications that can be introduced intonucleic acid molecules include, inosine, purine, pyridin-4-one,pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g.,5-methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine(e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.6-methyluridine), propyne, and others (Burgin et al., 1996,Biochemistry, 35, 14090; Uhlman & Peyman, supra). By “modified bases” inthis aspect is meant nucleotide bases other than adenine, guanine,cytosine and uracil at 1′ position or their equivalents; such bases maybe used at any position, for example, within the catalytic core of anenzymatic nucleic acid molecule and/or in the substrate-binding regionsof the nucleic acid molecule.

In a preferred embodiment, the invention features modified ribozymeswith phosphate backbone modifications comprising one or morephosphorothioate, phosphorodithioate, methylphosphonate, morpholino,amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate,sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl,substitutions. For a review of oligonucleotide backbone modificationssee Hunziker and Leumann, 1995, Nucleic Acid Analogues: Synthesis andProperties, in Modern Synthetic Methods, VCH, 331-417, and Mesmaeker etal., 1994, Novel Backbone Replacements for Oligonucleotides, inCarbohydrate Modifications in Antisense Research, ACS, 24-39. Thesereferences are hereby incorporated by reference herein.

By “abasic” is meant sugar moieties lacking a base or having otherchemical groups in place of a base at the 1′ position, (for moredetails, see Wincott et al., International PCT publication No. WO97/26270).

By “modified nucleoside” or “chemically modified” is meant anynucleotide which contains a modification in the chemical structure of anunmodified or naturally occurring nucleotide base, sugar and/orphosphate.

In connection with 2′-modified nucleotides as described for the presentinvention, by “amino” is meant 2′-NH₂ or 2′-O—NH₂, which may be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., WO98/28317, respectively, which are both incorporated by reference hereinin their entireties.

Various modifications to nucleic acid (e.g., antisense and ribozyme)structure can be made to enhance the utility of these molecules. Suchmodifications will enhance shelf-life, half-life in vitro, stability,and ease of introduction of such oligonucleotides to the target site,e.g., to enhance penetration of cellular membranes, and confer theability to recognize and bind to targeted cells.

Use of these molecules will lead to better treatment of the diseaseprogression by affording the possibility of combination therapies (e.g.,multiple ribozymes targeted to different genes, ribozymes coupled withknown small molecule inhibitors, or intermittent treatment withcombinations of ribozymes (including different ribozyme motifs) and/orother chemical or biological molecules). The treatment of patients withnucleic acid molecules may also include combinations of different typesof nucleic acid molecules. Therapies may be devised which include amixture of ribozymes (including different ribozyme motifs), antisenseand/or 2-5A chimera molecules to one or more targets to alleviatesymptoms of a disease.

Synthesis and Purification of Oligonucleotides Comprising One or MoreRibonucleotide

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs“small” refers to nucleic acid motifs no more than 100 nucleotides inlength, preferably no more than 80 nucleotides in length, and mostpreferably no more than 50 nucleotides in length; (e.g., individual siNAoligonucleotide sequences or siNA sequences synthesized in tandem) arepreferably used for exogenous delivery. The simple structure of thesemolecules increases the ability of the nucleic acid to invade targetedregions of protein and/or RNA structure. Exemplary molecules of theinstant invention are chemically synthesized, and others can similarlybe synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. All of these references areincorporated herein by reference. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 2.5 minute coupling step for 2′-O-methylated nucleotides and a 45second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoronucleotides. Table I outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 μmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol)of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyltetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycleof 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-foldexcess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-foldexcess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used ineach coupling cycle of deoxy residues relative to polymer-bound5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.synthesizer, determined by colorimetric quantitation of the tritylfractions, are typically 97.5-99%. Other oligonucleotide synthesisreagents for the 394 Applied Biosystems, Inc. synthesizer include thefollowing: detritylation solution is 3% TCA in methylene chloride (ABI);capping is performed with 16% N-methyl imidazole in THF (ABI) and 10%acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solutionis 16.9 mM I₂, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick &Jackson Synthesis Grade acetonitrile is used directly from the reagentbottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) is made upfrom the solid obtained from American International Chemical, Inc.Alternately, for the introduction of phosphorothioate linkages, Beaucagereagent (3H-1,2-Benzodithiol-3-one 1,1-dioxide, 0.05 M in acetonitrile)is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aqueousmethylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C.,the supernatant is removed from the polymer support. The support iswashed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and thesupernatant is then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder.

The method of synthesis used for RNA including certain siNA molecules ofthe invention follows the procedure as described in Usman et al., 1987,J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res.,18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5′-end, and phosphoramidites at the 3′-end. In a non-limitingexample, small scale syntheses are conducted on a 394 AppliedBiosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5minute coupling step for alkylsilyl protected nucleotides and a 2.5minute coupling step for 2′-O-methylated nucleotides. Table I outlinesthe amounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include the following:detritylation solution is 3% TCA in methylene chloride (ABI); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THF (ABI); oxidation solution is 16.9 mMI₂, 49 mM pyridine, 9% water in THF (PERSEPTIVE™). Burdick & JacksonSynthesis Grade acetonitrile is used directly from the reagent bottle.S-Ethyltetrazole solution (0.25 M in acetonitrile) is made up from thesolid obtained from American International Chemical, Inc. Alternately,for the introduction of phosphorothioate linkages, Beaucage reagent(3H-1,2-Benzodithiol-3-one 1,1-dioxide0.05 M in acetonitrile) is used.

Deprotection of the oligonucleotide comprising one or moreribonucleotides is performed according to the present invention.Oligonucleotides are purified according to the present invention, and/orby gel electrophoresis using general methods or are purified by highpressure liquid chromatography (HPLC; See Stinchcomb et al.,International PCT Publication No. WO 95/23225, the totality of which ishereby incorporated herein by reference) and are resuspended in water.For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 minutes. The cartridge is then washed again with water, saltexchanged with 1 M NaCl and washed with water again. The oligonucleotideis then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including but notlimited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

The nucleic acid molecules (e.g. siNA molecules) of the invention canalso be synthesized via a tandem synthesis methodology, wherein bothsiNA strands are synthesized as a single contiguous oligonucleotidefragment or strand separated by a cleavable linker which is subsequentlycleaved to provide separate siNA fragments or strands that hybridize andpermit purification of the siNA duplex (see McSwiggen et al., U.S. Ser.No. (10/444,853), filed May 23, 2003). The linker can be apolynucleotide linker or a non-nucleotide linker. The tandem synthesisof siNA as described herein can be readily adapted to bothmultiwell/multiplate synthesis platforms such as 96 well or similarlylarger multi-well platforms. The tandem synthesis of siNA as describedherein can also be readily adapted to large scale synthesis platformsemploying batch reactors, synthesis columns and the like.

A nucleic acid molecule (e.g. siNA molecule) can also be assembled fromtwo distinct nucleic acid strands or fragments wherein one fragmentincludes the sense region and the second fragment includes the antisenseregion of the RNA molecule.

The nucleic acid molecules of the present invention can be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purifiedby gel electrophoresis using general methods or can be purified by highpressure liquid chromatography (HPLC; see Wincott et al., supra, thetotality of which is hereby incorporated herein by reference) andre-suspended in water.

Deprotection of Oligonucleotides Comprising One or More Ribonucleotides

For large scale and high throughput chemical synthesis ofoligoribonucleotides, it is important that the two main steps involvedin the deprotection of oligoribonucleotides (i.e. basic treatment toremove amino protecting groups and phosphate protecting groups andfluoride treatment to remove the 2′-OH alkylsilyl protecting groups suchas the t-butyldimethylsilyl group) are condensed.

Stinchcomb et al., supra describe a time-efficient (approximately 2hours) one-pot deprotection protocol based on anhydrous methylamine andtriethylamine trihydrogen fluoride. Since it has been reported thatwater contamination during fluoride treatment may be detrimental to theefficiency of the desilylation reaction (Hogrefe et al, 1993, NucleicAcids Res., 21, 4739-4741), and since the use of aqueous methylamine incombination with TEA.3HF results in ribonucleotide degradation (seeExample 3), it has previously been thought necessary to use an anhydroussolution of base such as 33% methylamine in absolute ethanol followed byneat triethylamine trihydrofluoride to effectively deprotectoligoribonucleotides in a one-pot fashion. However, these conditionshave proven less than stellar for the complete deprotection of2′-N-phthaloyl protecting groups, as are used to protect the 2′-aminofunction of 2′-deoxy-2′-amino nucleoside containing nucleic acidmolecules since incomplete deprotection products result (see FIG. 2,compound B). Attempts to force the anhydrous deprotection reactionconditions with longer times and/or higher temperatures for the completeremoval of phthaloyl groups results in marked degradation of theribonucleotide species. Therefore, applicant investigated the use ofaqueous methylamine in conjunction with TEA.3HF and DMSO as a one potmethod for oligonucleotide deprotection. This method, surprisingly, doesnot cause the presumed alkaline hydrolysis of ribonucleotide linkageswhen used in the presence of DMSO. Application of the method withoutDMSO results in lower yields of full length nucleic acid, presumablyfrom alkaline hydrolysis of ribonucleotide linkages (see Example 3). Theone pot aqueous method described herein provides a significantly shortertime for oligonucleotide deprotection and provides material withincreased yield and purity when compared to existing two pot aqueous andone pot anhydrous methods.

EXAMPLES

The following are non-limiting examples showing the deprotection ofoligonucleotides.

Example 1 Small Scale Deprotection of an Oligonucleotide Comprising Oneor More Ribonucleotides with 2′-O-TBDMS/N-phthaloyl Protection Using aOne-pot Anhydrous Deprotection Method

A ribozyrne sequence (Table II) (200 μmole) containing two N-phthaloylprotected 2′-amino nucleosides was synthesized as described herein, onCPG support with a Pharmacia OPII synthesizer. After the synthesis, thesupport was dried for 15 to 30 min. Approximately 20 mg of the supportwas transferred to a 5 ml screw capped vial. A 1:1 mixture of 33%methylamine/ethanol (800 μl) and dry DMSO (800 μl) was added to thesupport and the mixture was heated at 65° C. using a heating block for15 min. The solution was cooled to rt and then filtered through a 0.5micron filter into another 5 ml screw capped vial. TEA.3HF (600 μl) wasadded to the reaction mixture followed by heating at 65° C. for 15 min.The mixture was then cooled and quenched with 50 mM NaOAc (2 ml). Thecorresponding deprotected, purified full length oligonucleotide wasanalyzed by Capillary Gel Electrophoresis and ES Mass Spec. The massspectrum revealed three peaks with masses corresponding to the fullydeprotected oligonucleotide, the oligonucleotide with one partiallycleaved phthaloyl group intact, and the oligonucleotide with twopartially cleaved phthaloyl groups intact (FIG. 3A). The CGEchromatograph indicated a single broad peak (FIG. 4A). A similarapproach can be utilized to deprotect other oligonucleotides, such assiRNA, antisense, and aptamer oligonucleotides.

Example 2 Small Scale Deprotection of an Oligonucleotide Comprising Oneor More Ribonucleotides with 2′-O-TBDMS/N-phthaloyl Protection Using aOne-pot Aqueous Deprotection Method

A ribozyme sequence (Table II) (200 μmole) was synthesized as describedherein on CPG support with a Pharmacia OPII synthesizer. After thesynthesis, the support was dried for 15 to 30 min. Approximately 20 mgof the support was transferred to a 5 ml screw capped vial and thesupport was heated with aqueous methylamine (1 ml) at 65° C. using aheating block for 15 min. The solution was cooled to rt and thenfiltered through a 0.5 micron filter into another 5 ml screw cappedvial. DMSO (1.6 ml) and TEA.3HF (600 μl) were added to the reactionmixture followed by heating at 65° C. for 15 min. The mixture was thencooled and quenched with 50 mM NaOAc (2 ml). The correspondingdeprotected, purified full-length oligonucleotide was analyzed byCapillary Gel Electrophoresis and ES Mass Spec. The mass spectrumrevealed one peak with a mass corresponding to the fully deprotectedoligonucleotide (FIG. 3B). The CGE chromatograph indicated a singlenarrow peak (FIG. 4B). A similarapproach can be utilized to deprotectother oligonucleotides, such as siRNA, antisense and aptameroligonucleotides containing ribonucleotides and/or having chemicalmodifications. Additionally, deprotection conditions can be modified toprovide the best possible yield and purity of the oligonucleotideconstructs. For example, applicant has observed that oligonucleotidescomprising 2′-deoxy-2′-fluoro nucleotides can degrade underinappropriate deprotection conditions. Such oligonucleotides aredeprotected using aqueous methylamine at about 35° C. for 30 minutes. Ifthe 2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

Example 3 Small Scale Deprotection of an Oligonucleotide Comprising Oneor More Ribonucleotides with 2′-O-TBDMS/N-phthaloyl Protection Using aOne-pot Aqueous Deprotection Method Without DMSO

A ribozyme sequence (Table II) (200 μmole) was synthesized as describedherein on CPG support with a Pharmacia OPII synthesizer. After thesynthesis, the support was dried for 15 to 30 min. Approximately 20 mgof the support was transferred to a 5 ml screw capped vial and thesupport was heated with aqueous methylamine (1 ml) at 65° C. using aheating block for 15 min. The solution was cooled to rt and thenfiltered through a 0.5 micron filter into another 5 ml screw cappedvial. TEA.3HF (600 μl) was added to the reaction mixture followed byheating at 65° C. for 15 min. The mixture was then cooled and quenchedwith 50 mM NaOAc (2 ml). The corresponding deprotected, purifiedfull-length oligonucleotide was analyzed by ion exchange HPLC. The HPLCtrace revealed significant degradation corresponding to cleavage ofribonucleotide linkages within the oligonucleotide when compared tomaterial from example 2 in which DMSO was used in the deprotection. Asimilar approach can be utilized to deprotect other oligonucleotides,such as siRNA, antisense and aptamer oligonucleotides containingribonucleotides and/or having chemical modifications. Additionally,deprotection conditions can be modified to provide the best possibleyield and purity of the oligonucleotide constructs. For example,applicant has observed that oligonucleotides comprising2′-deoxy-2′-fluoro nucleotides can degrade under inappropriatedeprotection conditions. Such oligonucleotides are deprotected usingaqueous methylamine at about 35° C. for 30 minutes. If the2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

Example 4 Large Scale Deprotection of an Oligonucleotide Comprising Oneor More Ribonucleotides with 2′-O-TBDMS Protection Using a One-potAnhydrous Deprotection Method

A ribozyme sequence (Table II) (200 μmole) was synthesized as describedherein on CPG support with a Pharmacia OPII synthesizer. After thesynthesis, the support was dried for 15 to 30 min and transferred to a500 ml Schott bottle. A 1:1 mixture of 33% methylamine/ethanol (75 ml)and dry DMSO (75 ml) was added to the support and the mixture was heatedat 35° C. in an incubated shaker for 4 h. The solution was cooled to rt(15 min) and then filtered through a sintered glass funnel. The supportwas washed with DMSO (4×15 ml) and the combined filtrate was cooled inan ice bath for 30 min. TEA.3HF (30 ml) was added to the reactionmixture followed by heating at 65° C. for 1 h. The mixture was thencooled at −78° C. for 30 min and quenched with 50 mM NaOAc (200 ml). Asimilar approach can be utilized to deprotect other oligonucleotides,such as siRNA, antisense, and apatmer oligonucleotides.

Example 5 Large Scale Deprotection of an Oligonucleotide Comprising Oneor More Ribonucleotides with 2′-O-TBDMS Protection Using a One-potAqueous Deprotection Method

A ribozyme sequence (Table II) (200 μmole) was synthesized describedherein on CPG support with a Pharmacia OPII synthesizer. After thesynthesis, the support was dried for 15 to 30 min and transferred to a250 ml Schott bottle. 40% Aqueous methylamine (75 ml) was added to thesupport and the mixture was heated at 35° C. in an incubated shaker for1 h. The solution was cooled to rt (15 min) and then filtered through asintered glass funnel. The support was washed with DMSO (4×18.75 ml) andthe combined filtrate was cooled in an ice bath for 30 min. TEA.3HF (45ml) was added to the reaction mixture followed by heating at 65° C. for1 h. The mixture was then cooled at −78° C. for 30 min and quenched with50 mM NaOAc (195 ml). A similarapproach can be utilized to deprotectother oligonucleotides, such as siRNA, antisense and aptameroligonucleotides containing ribonucleotides and/or having chemicalmodifications. Additionally, deprotection conditions can be modified toprovide the best possible yield and purity of the oligonucleotideconstructs. For example, applicant has observed that oligonucleotidescomprising 2′-deoxy-2′-fluoro nucleotides can degrade underinappropriate deprotection conditions. Such oligonucleotides aredeprotected using aqueous methylamine at about 35° C. for 30 minutes. Ifthe 2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

Example 6 Large Scale Deprotection of an Oligonucleotide Comprising Oneor More Ribonucleotides with 2′-O-TBDMS/N-phthaloyl Protection Using aOne-pot Aqueous Deprotection Method

A ribozyme sequence (Table II) (200 μmole) was synthesized as describedherein on CPG support with a Pharmacia OPII synthesizer. After thesynthesis, the support was dried for 15 to 30 min and transferred to a250 ml Schott bottle. 40% Aqueous methylamine (75 ml) was added to thesupport and the mixture was heated at 65° C. in an incubated shaker for1 h. The solution was cooled to rt (15 min) and then filtered through asintered glass funnel. The support was washed with DMSO (4×18.75 ml) andthe combined filtrate was cooled at −78° C. for 30 min. TEA.3HF (45 ml)was added to the reaction mixture followed by heating at 65° C. for 1 h.The mixture was then cooled in an ice bath for 30 min and quenched with50 mM NaOAc (195 ml). A similarapproach can be utilized to deprotectother oligonucleotides, such as siRNA, antisense and aptameroligonucleotides containing ribonucleotides and/or having chemicalmodifications. Additionally, deprotection conditions can be modified toprovide the best possible yield and purity of the oligonucleotideconstructs. For example, applicant has observed that oligonucleotidescomprising 2′-deoxy-2′-fluoro nucleotides can degrade underinappropriate deprotection conditions. Such oligonucleotides aredeprotected using aqueous methylamine at about 35° C. for 30 minutes. Ifthe 2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes.

Example 7 Large Scale Ion Exchange Purification of an OligonucleotideComprising One or More Ribonucleotidess

Oligonucleotide comprising one or more ribonucleotides (e.g., siRNA,antisense, or aptamer oligonucleotides) are purified by ion exchangechromatography following deprotection. The ion-exchange purificationprocess can be performed on both Pharmacia Source Q15 and BioradMacroprep 25Q type media. The buffer used for equilibration of thepurification media is either 0-20% ethanol (200 proof USP grade) oracetonitrile, in 20 mmolar sodium phosphate and 0.1 M NaCl. The samebuffer can be used for loading the nucleic acid molecule onto thepurification media, or alternatively, water can be used. The crudeoligonucleotide material is loaded on the column in concentrations up to10 mg/mL. Application of a suitable gradient of an elution buffer suchas 1.0 M NaCl can be used to isolate fractions. Following purification,the fractions are analyzed for purity by a suitable method (for exampleUV, HPLC and/or CGE). The pure fractions are pooled and desalting isperformed via tangential flow filtration using membranes such asSartorius or Pall Filtron PES 1 K membranes. The concentrated materialis then lyophilized. A similar approach can be used to purify siRNAconstructs of the invention, such as single stranded, haipin, and duplexsiRNA. For duplex siRNA, each strand is synthesized, deprotected, andpurified separately, then hybridized under conditions suitable forduplex formation. In a non-limiting example, siRNA strands are annealedin 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesiumacetate at 20 μM each strand. The annealing mixture is first heated to90° C. for 1 minute and then is transferred to 37° C. for 60 minutes.Annealing is confirmed by non-denaturing PAGE. Alternately, duplex siRNAcan be synthesized using a tandem synthesis approach as described inExample 8 below.

Example 8 Tandem Synthesis of siRNA Constructs

Exemplary siRNA molecules of the invention are synthesized in tandemusing a cleavable linker, for example, a succinyl-based linker. Tandemsynthesis as described herein is followed by a one-step purificationprocess that provides RNAi molecules in high yield. This approach ishighly amenable to siNA synthesis in support of high throughput RNAiscreening, and can be readily adapted to multi-column or multi-wellsynthesis platforms.

After completing a tandem synthesis of a siNA oligo and its complementin which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact(trityl on synthesis), the oligonucleotides are deprotected as describedabove, for example as described in Example 2. Following deprotection,the siNA sequence strands are allowed to spontaneously hybridize. Thishybridization yields a duplex in which one strand has retained the5′-O-DMT group while the complementary strand comprises a terminal5′-hydroxyl. The newly formed duplex behaves as a single molecule duringroutine solid-phase extraction purification (Trityl-On purification)even though only one molecule has a dimethoxytrityl group. Because thestrands form a stable duplex, this dimethoxytrityl group (or anequivalent group, such as other trityl groups or other hydrophobicmoieties) is all that is required to purify the pair of oligos, forexample, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point ofintroducing a tandem linker, such as an inverted deoxy abasic succinateor glyceryl succinate linker (see FIG. 5) or an equivalent cleavablelinker. A non-limiting example of linker coupling conditions that can beused includes a hindered base such as diisopropylethylamine (DIPA)and/or DMAP in the presence of an activator reagent such asBromotripyrrolidinophosphoniumhexaflurorophosphate (PyBrOP). After thelinker is coupled, standard synthesis chemistry is utilized to completesynthesis of the second sequence leaving the terminal the 5′-O-DMTintact. Following synthesis, the resulting oligonucleotide isdeprotected according to the procedures described herein and quenchedwith a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.

Purification of the siRNA duplex can be readily accomplished using solidphase extraction, for example using a Waters C18 SepPak 1 g cartridgeconditioned with 1 column volume (CV) of acetonitrile, 2 CV H2O, and 2CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H2O or 50mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with1 CV H2O followed by on-column detritylation, for example by passing 1CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then addinga second CV of 1% aqueous TFA to the column and allowing to stand forapproximately 10 minutes. The remaining TFA solution is removed and thecolumn washed with H2O followed by 1 CV 1M NaCl and additional H2O. ThesiNA duplex product is then eluted, for example, using 1 CV 20% aqueousCAN.

FIG. 6 provides an example of MALDI-TOF mass spectrometry analysis of apurified siNA construct in which each peak corresponds to the calculatedmass of an individual siNA strand of the siNA duplex. The same purifiedsiNA provides three peaks when analyzed by capillary gel electrophoresis(CGE), one peak presumably corresponding to the duplex siNA, and twopeaks presumably corresponding to the separate siNA sequence strands.Ion exchange HPLC analysis of the same siNA contract only shows a singlepeak. Testing of the purified siNA construct using a luciferase reporterassay described below demonstrated the same RNAi activity compared tosiNA constructs generated from separately synthesized oligonucleotidesequence strands.

Other Uses

The nucleic acid molecules of this invention (e.g., ribozymes) may beused as therapeutic agents to treat a broad spectrum of diseases andconditions. Ribozymes are RNA molecules having an enzymatic activitywhich is able to repeatedly cleave other separate RNA molecules in anucleotide base sequence specific manner. Such enzymatic RNA moleculescan be targeted to virtually any RNA transcript, and efficient cleavageachieved in vitro. Kim et al., 1987, Proc. Nat. Acad. of Sci. USA, 84,8788, Hazeloff et al., 1988 Nature, 234, 585, Cech, 1988, JAMA, 260,3030, and Jefferies et al., 1989, Nucleic Acid Research, 17, 1371.Ribozymes act by first binding to a target RNA. Such binding occursthrough the target RNA binding portion of a ribozyme which is held inclose proximity to an enzymatic portion of the RNA which acts to cleavethe target RNA. Thus, the ribozyme first recognizes and then binds atarget RNA through complementary base-pairing, and once bound to thecorrect site, acts enzymatically to cut the target RNA. Strategiccleavage of such a target RNA will destroy its ability to directsynthesis of an encoded protein. After a ribozyme has bound and cleavedits RNA target it is released from that RNA to search for another targetand can repeatedly bind and cleave new targets.

The nucleic acid molecules of the invention may be used as diagnostictools to examine genetic drift and mutations within diseased cells or todetect the presence of a particular RNA in a cell. The closerelationship between ribozyme activity and the structure of the targetRNA allows the detection of mutations in any region of the moleculewhich alters the base-pairing and three-dimensional structure of thetarget RNA. By using multiple ribozymes described in this invention, onemay map nucleotide changes which are important to RNA structure andfunction in vitro, as well as in cells and tissues. Cleavage of targetRNAs with ribozymes may be used to inhibit gene expression and definethe role (essentially) of specified gene products in the progression ofdisease. In this manner, other genetic targets may be defined asimportant mediators of the disease. These experiments will lead tobetter treatment of disease progression by affording the possibility ofcombinational therapies (e.g., multiple ribozymes targeted to differentgenes, ribozymes coupled with known small molecule inhibitors, orintermittent treatment with combinations of ribozymes and/or otherchemical or biological molecules). Other in vitro uses of ribozymes ofthis invention are well known in the art, and include detection of thepresence of mRNAs associated with a RNA-related condition. Such RNA isdetected by determining the presence of a cleavage product aftertreatment with a ribozyme using standard methodology.

In a specific example, ribozymes which can cleave only wild-type ormutant forms of the target RNA are used for the assay. The firstribozyme is used to identify wild-type RNA present in the sample and thesecond ribozyme will be used to identify mutant RNA in the sample. Asreaction controls, synthetic substrates of both wild-type and mutant RNAwill be cleaved by both ribozymes to demonstrate the relative ribozymeefficiencies in the reactions and the absence of cleavage of the“non-targeted” RNA species. The cleavage products from the syntheticsubstrates will also serve to generate size markers for the analysis ofwild-type and mutant RNAs in the sample population. Thus, each analysiscan require two ribozymes, two substrates and one unknown sample, whichwill be combined into six reactions. The presence of cleavage productswill be determined using an RNAse protection assay so that full-lengthand cleavage fragments of each RNA can be analyzed in one lane of apolyacrylamide gel. It is not absolutely required to quantify theresults to gain insight into the expression of mutant RNAs and putativerisk of the desired phenotypic changes in target cells. The expressionof mRNA whose protein product is implicated in the development of thephenotype is adequate to establish risk. If probes of comparablespecific activity are used for both transcripts, then a qualitativecomparison of RNA levels will be adequate and will decrease the cost ofthe initial diagnosis. Higher mutant form to wild-type ratios will becorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications may be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims.

The invention illustratively described herein suitably may be practicedin the absence of any element or elements, limitation or limitationswhich is not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed may be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

Thus, additional embodiments are within the scope of the invention andwithin the following claims:

Other embodiments are within the following claims. TABLE I ReagentEquivalents Amount Wait Time* DNA Wait Time* 2′-O-methyl Wait Time* RNAA. 2.5 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites 6.5 163μL 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole 23.8 238 μL 45 sec 2.5 min7.5 min Acetic Anhydride 100 233 μL 5 sec 5 sec 5 sec N-Methyl 186 233μL 5 sec 5 sec 5 sec Imidazole TCA 176 2.3 mL 21 sec 21 sec 21 secIodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage 12.9 645 μL 100 sec 300sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B. 0.2 μmol Synthesis CycleABI 394 Instrument Phosphoramidites 15 31 μL 45 sec 233 sec 465 secS-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min 465 sec Acetic Anhydride 655124 μL 5 sec 5 sec 5 sec N-Methyl 1245 124 μL 5 sec 5 sec 5 secImidazole TCA 700 732 μL 10 sec 10 sec 10 sec Iodine 20.6 244 μL 15 sec15 sec 15 sec Beaucage 7.7 232 μL 100 sec 300 sec 300 sec AcetonitrileNA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle 96 well InstrumentEquivalents: DNA/ Amount: DNA/2′-O- Wait Time* Reagent 2′-O-methyl/Ribomethyl/Ribo DNA Wait Time* 2′-O-methyl Wait Time* Ribo Phosphoramidites22/33/66 40/60/120 μL 60 sec 180 sec 360 sec S-Ethyl Tetrazole70/105/210 40/60/120 μL 60 sec 180 min 360 sec Acetic Anhydride265/265/265 50/50/50 μL 10 sec 10 sec 10 sec N-Methyl 502/502/50250/50/50 μL 10 sec 10 sec 10 sec Imidazole TCA 238/475/475 250/500/500μL 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30 sec 30 sec 30sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 sec Acetonitrile NA1150/1150/1150 μL NA NA NA*Wait time does not include contact time during delivery.

TABLE II Nucleic Acid Sequence used in Deprotection Studies Com- Seqpound ID Sequence No. No. g_(s)c_(s)a_(s)g_(s)ug GccgaaagGCGaGuGaGGuCuagcuca B 19292 1Lower case = 2′-O-methylUpper Case = ribonucleotideC = 2′-deoxy-2′-amino Cytidines = phosphorothioate internucleotide linkageB = inverted deoxy abasic moiety

TABLE III Non-limiting examples of Stabilization Chemistries forchemically modified siNA constructs Chemistry pyrimidine Purine cap p =S Strand “Stab 00” Ribo Ribo TT at 3′- S/AS ends “Stab 1” Ribo Ribo — 5at 5′-end S/AS 1 at 3′-end “Stab 2” Ribo Ribo — All Usually linkages AS“Stab 3” 2′-fluoro Ribo — 4 at 5′-end Usually S 4 at 3′-end “Stab 4”2′-fluoro Ribo 5′ and 3′- — Usually S ends “Stab 5” 2′-fluoro Ribo — 1at 3′-end Usually AS “Stab 6” 2′-O- Ribo 5′ and 3′- — Usually S Methylends “Stab 7” 2′-fluoro 2′-deoxy 5′ and 3′- — Usually S ends “Stab 8”2′-fluoro 2′-O- — 1 at 3′-end S/AS Methyl “Stab 9” Ribo Ribo 5′ and 3′-— Usually S ends “Stab 10” Ribo Ribo — 1 at 3′-end Usually AS “Stab 11”2′-fluoro 2′-deoxy — 1 at 3′-end Usually AS “Stab 12” 2′-fluoro LNA 5′and 3′- Usually S ends “Stab 13” 2′-fluoro LNA 1 at 3′-end Usually AS“Stab 14” 2′-fluoro 2′-deoxy 2 at 5′-end Usually 1 at 3′-end AS “Stab15” 2′-deoxy 2′-deoxy 2 at 5′-end Usually 1 at 3′-end AS “Stab 16” Ribo2′-O- 5′ and 3′- Usually S Methyl ends “Stab 17” 2′-O- 2′-O- 5′ and 3′-Usually S Methyl Methyl ends “Stab 18” 2′-fluoro 2′-O- 5′ and 3′-Usually S Methyl ends “Stab 19” 2′-fluoro 2′-O- 3′-end S/AS Methyl “Stab20” 2′-fluoro 2′-deoxy 3′-end Usually AS “Stab 21” 2′-fluoro Ribo 3′-endUsually AS “Stab 22” Ribo Ribo 3′-end Usually AS “Stab 23” 2′-fluoro*2′-deoxy* 5′ and 3′- Usually S ends “Stab 24” 2′-fluoro* 2′-O- — 1 at3′-end S/AS Methyl* “Stab 25” 2′-fluoro* 2′-O- — 1 at 3′-end S/ASMethyl* “Stab 26” 2′-fluoro* 2′-O- — S/AS Methyl* “Stab 27” 2′-fluoro*2′-O- 3′-end S/AS Methyl* “Stab 28” 2′-fluoro* 2′-O- 3′-end S/AS Methyl*“Stab 29” 2′-fluoro* 2′-O- 1 at 3′-end S/AS Methyl* “Stab 30” 2′-fluoro*2′-O- S/AS Methyl* “Stab 31” 2′-fluoro* 2′-O- 3′-end S/AS Methyl* “Stab32” 2′-fluoro 2′-O- S/AS Methyl “Stab 33” 2′-fluoro 2′-deoxy* 5′ and 3′-— Usually S ends “Stab 34” 2′-fluoro 2′-O- 5′ and 3′- Usually S Methyl*ends “Stab 3F” 2′-OCF3 Ribo — 4 at 5′-end Usually S 4 at 3′-end “Stab4F” 2′-OCF3 Ribo 5′ and 3′- — Usually S ends “Stab 5F” 2′-OCF3 Ribo — 1at 3′-end Usually AS “Stab 7F” 2′-OCF3 2′-deoxy 5′ and 3′- — Usually Sends “Stab 8F” 2′-OCF3 2′-O- — 1 at 3′-end S/AS Methyl “Stab 11F”2′-OCF3 2′-deoxy — 1 at 3′-end Usually AS “Stab 12F” 2′-OCF3 LNA 5′ and3′- Usually S ends “Stab 13F” 2′-OCF3 LNA 1 at 3′-end Usually AS “Stab14F” 2′-OCF3 2′-deoxy 2 at 5′-end Usually 1 at 3′-end AS “Stab 15F”2′-OCF3 2′-deoxy 2 at 5′-end Usually 1 at 3′-end AS “Stab 18F” 2′-OCF32′-O- 5′ and 3′- Usually S Methyl ends “Stab 19F” 2′-OCF3 2′-O- 3′-endS/AS Methyl “Stab 20F” 2′-OCF3 2′-deoxy 3′-end Usually AS “Stab 21F”2′-OCF3 Ribo 3′-end Usually AS “Stab 23F” 2′-OCF3* 2′-deoxy* 5′ and 3′-Usually S ends “Stab 24F” 2′-OCF3* 2′-O- — 1 at 3′-end S/AS Methyl*“Stab 25F” 2′-OCF3* 2′-O- — 1 at 3′-end S/AS Methyl* “Stab 26F” 2′-OCF3*2′-O- — S/AS Methyl* “Stab 27F” 2′-OCF3* 2′-O- 3′-end S/AS Methyl* “Stab28F” 2′-OCF3* 2′-O- 3′-end S/AS Methyl* “Stab 29F” 2′-OCF3* 2′-O- 1 at3′-end S/AS Methyl* “Stab 30F” 2′-OCF3* 2′-O- S/AS Methyl* “Stab 31F”2′-OCF3* 2′-O- 3′-end S/AS Methyl* “Stab 32F” 2′-OCF3 2′-O- S/AS Methyl“Stab 33F” 2′-OCF3 2′-deoxy* 5′ and 3′- — Usually S ends “Stab 34F”2′-OCF3 2′-O- 5′ and 3′- Usually S Methyl* endsCAP = any terminal cap, see for example FIG. 10.All Stab 00-34 chemistries can comprise 3′-terminal thymidine (TT)residuesAll Stab 00-34 chemistries typically comprise about 21 nucleotides, butcan vary as described herein.S = sense strandAS = antisense strand*Stab 23 has a single ribonucleotide adjacent to 3′-CAP*Stab 24 and Stab 28 have a single ribonucleotide at 5′-terminus*Stab 25, Stab 26, and Stab 27 have three ribonucleotides at 5′-terminus*Stab 29, Stab 30, Stab 31, Stab 33, and Stab 34 any purine at firstthree nucleotide positions from 5′-terminus are ribonucleotidesp = phosphorothioate linkage

1. A process comprising the steps of: a) synthesizing a nucleic acidmolecule comprising one or more ribonucleotides, using a method selectedfrom the group consisting of solid phase phosphoramidite, solution phasephosphoramidite, solid phase H-phosphonate, solution phaseH-phosphonate, hybrid phase phosphoramidite, and hybrid phaseH-phosphonate-based synthetic methods; b) contacting said nucleic acidmolecule from step (a) with aqueous alkylamine, trialkylamine, oralkylamine and trialkylamine, under conditions suitable for the removalof any 2′-amino protecting groups, exocyclic amino (base) protectinggroups and/or phosphate protecting groups, which may be individuallypresent or absent, from said molecule; c) contacting reaction mixturehaving said nucleic acid molecule from step (b) with a polar solvent andtrialkylamine.hydrogen fluoride under conditions suitable for theremoval of a 2′-OH protecting group; d) loading reaction mixture havingsaid nucleic acid molecule from step (c) onto a media selected from thegroup consisting of Pharmacia Source Q15, Biorad Macroprep 25Q,Pharmacia Q-sepharose, Perceptive POROS HQ, TOSOHAAS Q-5PW-HR, Q-5PW,and super Q-5PW media in a suitable buffer comprising buffers selectedfrom the group consisting of water, 20% ethanol in about 20 mM sodiumphosphate and about 0.1 M NaCl and acetonitrile in about 20 mM sodiumphosphate and about 0.1 M NaCl; e) applying a purification gradientusing a suitable elution buffer, analyzing the fractions and allowingfor the pure fractions to be pooled and desalted.
 2. The process ofclaim 1, wherein said nucleic acid molecule comprising one or moreribonucleotides is a siRNA molecule.
 3. The process of claim 2, whereinsaid siRNA molecule further comprises one or more 2′-deoxy-2′-fluoronucleotides.
 4. The process of claim 1, wherein said aqueous alkylamineis aqueous methylamine.
 5. The process of claim 1, wherein said aqueousalkylamine is 40% aqueous methylamine.
 6. The process of claim 1,wherein said trialkylamine.trihydrofluoride istrialkylamine.trihydrofluoride (TEA.3HF).
 7. The process of claim 1,wherein said 2′-OH protecting group comprises the t-butyldimethylsilyl(TBDMSi) protecting group and derivatives thereof.
 8. The process ofclaim 1, wherein said nucleic acid molecule comprises one or morechemical modifications.
 9. A process of purifying a nucleic acidmolecule, comprising the steps of: a) loading said nucleic acid moleculeonto a media selected from the group consisting of Pharmacia Source Q15,Biorad Macroprep 25Q, Pharmacia Q-sepharose, Perceptive POROS HQ,TOSOHAAS Q-5PW-HR, Q-5PW, and super Q-5PW media in a loading buffercomprising buffers selected from the group consisting of water, 20%ethanol in about 20 mM sodium phosphate and about 0.1 M NaCl andacetonitrile in about 20 mM sodium phosphate and about 0.1 M NaCl; b)applying a purification gradient using a suitable elution buffer,analyzing the fractions and allowing for the pure fractions to be pooledand desalted.
 10. The process of claim 10, wherein said nucleic acidmolecule comprises one or more ribonucleotides.
 11. The process of claim10, wherein said nucleic acid molecule comprises one or more chemicalmodifications.
 12. The process of claims 1 or 10, wherein said nucleicacid molecule is a single stranded nucleic acid molecule.
 13. Theprocess of claims 1 or 10, wherein said nucleic acid molecule is adouble stranded nucleic acid molecule.
 14. The process of claim 13,wherein said double stranded nucleic acid molecule comprises one or morechemical modifications.
 15. The process of claim 13, wherein said eachstrand of the double stranded nucleic acid molecule is of length between19 and 23 nucleotides.
 16. The process of claim 12, wherein said eachstrand of the double stranded nucleic acid molecule is of length between19 and 23 nucleotides.
 17. The process of claims 8 or 11, wherein saidchemical modification is a sugar modification.
 18. The process of claim17, wherein said sugar modification is a 2′-sugar modification.
 19. Theprocess of claims 8 or 11, wherein said chemical modification is a basemodification.
 20. The process of claims 8 or 11, wherein said chemicalmodification is a phosphate backbone modification.
 21. The process ofclaim 20, wherein said phosphate backbone modification isphosphorothioate.
 22. The process of claims 8 or 11, wherein saidchemical modification is a terminal end modification.
 23. The process ofclaim 22, wherein said end modification is at the 5′-end of said nucleicacid molecule.
 24. The process of claim 22, wherein said endmodification is at the 3′-end of said nucleic acid molecule.
 25. Theprocess of claim 22, wherein said end modification is at both the 5′-and 3′-end of said nucleic acid molecule.